Compositions and methods for inhibiting arginase activity

ABSTRACT

The invention relates to a novel class of compounds that exhibit activity inhibitory activity toward arginase, and pharmaceutical compositions comprising the compounds of the invention. Also provided herein are methods of treating cancer with the arginase inhibitors of the invention.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application No. 62/248,632, filed Oct. 30, 2015; U.S. ProvisionalPatent Application No. 62/281,964, filed Jan. 22, 2016; and U.S.Provisional Patent Application No. 62/323,034, filed Apr. 15, 2016,which applications are hereby incorporated by reference in theirentirety.

BACKGROUND

Cancer is characterized by the uncontrolled growth of cells in the body,leading to the invasion of essential organs and often death. Initially,the pharmacological treatment of cancer utilized non-specific cytotoxicagents that targeted all rapidly dividing cells, including normal cells.These non-specific cytotoxic agents have anti-tumor effects but theiruse is often limited by severe toxicities. As the understanding of theproteins and pathways that enable cancer cells to thrive has evolved,newer more targeted agents have been developed that block specificproteins that are activated in cancer cells.

An emerging field for the development of therapeutics that addresses thechallenges presented in treating cancers is immuno-oncology, alsoreferred to as tumor immunology. Certain tumor types have developedmechanisms to escape destruction by the body's immune system. Tumorimmunology is a therapeutic area focused on activating the body's ownimmune system to attack and kill tumors. The naturally occurring aminoacid arginine is implicated in tumor immunology, as it is important forthe activation, growth, and survival of a body's cancer-fightingcytotoxic T-cells.

However, levels of arginine are depleted in the tumor microenvironmentby arginase, an enzyme produced and secreted by neutrophils and myeloidderived suppressor cells (MDSCs) that accumulate in cancer patients ofmultiple histotypes. In fact, elevated levels of arginase enzyme havebeen observed in the plasma of renal cell carcinoma, breast cancer,chronic myelogenous leukemia, esophageal cancer, prostate cancer,non-small cell lung cancer, glioblastoma, and acute myeloid leukemiapatients. Therefore, there is a need to develop inhibitors of arginasethat restore arginine levels in the tumor microenvironment, thereforepromoting the tumor-killing activity of cytotoxic T-cells.

SUMMARY OF INVENTION

In certain embodiments, the invention provides a series of novelarginase inhibitor compounds. The compounds of the invention have astructure of formula (I):

-   -   or a pharmaceutically acceptable salt or prodrug thereof;    -   wherein:    -   R^(a) is H or is selected from optionally substituted alkyl,        alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,        (cycloalkyl)alkyl, (heterocycloalkyl)alkyl, aryl, heteroaryl,        aralkyl, and heteroaralkyl;    -   R^(b) is H or is selected from optionally substituted alkyl,        alkenyl, alkynyl, acyl, —C(O)O(alkyl), and —C(O)O(aryl);    -   each R^(c) is independently selected from H or alkyl, or two        occurrences of R^(c) are taken together with the intervening        —O—B—O— atoms to form an optionally substituted boron-containing        ring;    -   X is O or S;    -   R¹ and R² are each independently selected from H and optionally        substituted alkyl, alkenyl, alkynyl, cycloalkyl,        (cycloalkyl)alkyl, heterocycloalkyl, (heterocycloalkyl)alkyl,        aryl, heteroaryl, aralkyl, and heteroaralkyl;    -   or R¹ and R² are taken together with the intervening atoms to        form an optionally substituted 5- to 7-membered ring; and    -   R³ is H or optionally substituted alkyl;    -   or R¹ and R³ are taken together with the intervening atoms to        form an optionally substituted 5- to 7-membered ring;    -   wherein the compound is not:

In certain embodiments, the invention also provides pharmaceuticalcompositions comprising a compound of the invention and apharmaceutically acceptable carrier.

In certain embodiments, the invention provides methods of treating orpreventing cancer, comprising administering to a subject in need thereofa therapeutically effective amount of a compound or pharmaceuticalcomposition of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the tumor volume over time. Arginaseinhibitor Compound 10, administered as a single agent, slows tumorgrowth relative to control in mice implanted with Lewis Lung Carcinomacells.

FIG. 2 is a graph depicting the tumor volume over time. Madison109murine lung carcinoma cells were implanted in balb/c mice and mice weredosed orally with vehicle or arginase inhibitor Compound 10 BID (N=10per group).

FIG. 3 is a graph depicting the tumor volume overtime. B16F10 murinemelanoma cells were implanted in C57.Bl/6 mice and mice were dosedorally with vehicle or arginase inhibitor Compound 10 BID (N=10 pergroup).

FIG. 4 consists of panels A and B, and depicts the growth of 4T1 mammarycarcinoma cells implanted orthotopically into female balb/c mice andtreated with either vehicle; Compound 10 (100 mg/kg PO BID); anti-CTLA-4(5 mg/kg IP on Days 2, 5, 8) plus anti-PD-1 (5 mg/kg IP on days 3, 6,and 9); or the combination of Compound 10 with anti-CTLA-4 and anti-PD-1(N=10 per group; *P<0.05; ***P<0.001, **** P<0.0001 vs vehicle).

FIG. 5 is a graph depicting the tumor volume over time. Female C57.Bl/6mice were implanted subcutaneously with 1×10⁶ B16.F10 murine melanomacells. On Day 2, mice were randomized into the following groups of n=10mice; 1) Vehicle PO BID; 2) Compound 10, 100 mg/kg PO BID; 3)Epacadostat, 100 mg/kg PO BID; or 4) Compound 10 and Epacadostat (100mg/kg PO BID each). Tumors were measured with calipers three times perweek and tumor volume calculated using the formula tumor volume(mm³)=(a×b²/2) where ‘b’ is the smallest diameter and ‘a’ is the largestperpendicular diameter. *P-value <0.05 (ANOVA).

FIG. 6 is a graph depicting the tumor volume over time. Female balb/cmice were implanted subcutaneously with 1×10⁶ CT26 murine coloncarcinoma cells. On Day 2, mice were randomized into the followinggroups of n=10 mice; 1) Vehicle PO BID starting day 2; 2) Compound 10,100 mg/kg PO BID starting day 2; 3) Gemcitabine, 50 mg/kg IP on days 10and 16; or 4) Compound 10 and Gemcitabine at their respective regimens.Tumors were measured with calipers three times per week and tumor volumecalculated using the formula tumor volume (mm³)=(a×b²/2) where ‘b’ isthe smallest diameter and ‘a’ is the largest perpendicular diameter.*P-value <0.05 (ANOVA).

FIG. 7 is a graph depicting the tumor volume over time. Female balb/cmice were implanted subcutaneously with 1×10⁶ CT26 murine coloncarcinoma cells. On Day 2, mice were randomized into the followinggroups of n=10 mice; 1) Vehicle PO BID; 2) Compound 10, 100 mg/kg POBID; 3) anti-PD-L1 (clone 10f.9g2), 5 mg/kg IP on days 5, 7, 9, 11, 13,and 15; or 4) Compound 10 and anti-PD-L1. Tumors were measured withcalipers three times per week and tumor volume calculated using theformula tumor volume (mm³)=(a×b²/2) where ‘b’ is the smallest diameterand ‘a’ is the largest perpendicular diameter.

FIG. 8 depicts the percent survival over time. Female balb/c mice wereimplanted subcutaneously with 5×10⁴ Madison 109 murine lung carcinomacells. On Day 2, mice were randomized into the following groups of n=10mice; 1) Vehicle PO BID; 2) Compound 10, 100 mg/kg PO BID; 3) Whole bodyradiation (X-ray) 2 gy on Days 10-14 and days 17-21; or 4) Compound 10and radiation. Tumors were measured with calipers two times per week andtumor volume calculated using the formula tumor volume (mm³)=(a×b²/2)where ‘b’ is the smallest diameter and ‘a’ is the largest perpendiculardiameter. *P value <0.05 (log-rank test).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides small molecule inhibitors of arginase.

Compounds of the Invention

In certain embodiments, the invention provides a compound having astructure of formula (I):

-   -   or a pharmaceutically acceptable salt or prodrug thereof;    -   wherein:    -   R^(a) is H or is selected from optionally substituted alkyl,        alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,        (cycloalkyl)alkyl, (heterocycloalkyl)alkyl, aryl, heteroaryl,        aralkyl, and heteroaralkyl;    -   R^(b) is H or is selected from optionally substituted alkyl,        alkenyl, alkynyl, acyl, —C(O)O(alkyl), and —C(O)O(aryl);    -   each R^(c) is independently selected from H or alkyl, or two        occurrences of R^(c) are taken together with the intervening        —O—B—O— atoms to form an optionally substituted boron-containing        ring;    -   X is O or S;    -   R¹ and R² are each independently selected from H and optionally        substituted alkyl, alkenyl, alkynyl, cycloalkyl,        (cycloalkyl)alkyl, heterocycloalkyl, (heterocycloalkyl)alkyl,        aryl, heteroaryl, aralkyl, and heteroaralkyl; or    -   R¹ and R² are taken together with the intervening atoms to form        an optionally substituted 5- to 7-membered ring; and    -   R³ is H or optionally substituted alkyl;    -   or R¹ and R³ are taken together with the intervening atoms to        form an optionally substituted 5- to 7-membered ring;    -   wherein the compound is not:

In certain embodiments, the compound of formula (I) has a structure offormula (Ia):

In certain embodiments, the compound of formula I as a structure offormula (Ib):

In certain embodiments, the compound of formula (I) has a structure offormula (Ic):

In certain embodiments, the compound of formula (I) has a structure offormula (Id):

In certain embodiments, the compound of formula (I) has a structure offormula (Ie):

In certain embodiments, the compound of formula (I) has a structure offormula (If):

In certain embodiments, the compound of formula (I) has a structure offormula (Ig):

In certain embodiments, the compound of formula (I) has a structure offormula

In certain embodiments of any of formulae (I), (Ia), and (Ib), R² is H.

In certain embodiments of any of the foregoing formulae, R^(a) is H oroptionally substituted alkyl. In certain preferred embodiments, R^(a) isH.

In certain embodiments of any of the foregoing formulae, R^(b) is H oroptionally substituted alkyl or acyl. In certain preferred embodiments,R^(b) is H.

In certain embodiments of any of the foregoing formulae, R^(c) is H foreach occurrence.

In certain embodiments of any of the foregoing formulae, two occurrencesof R^(c) are taken together to form an optionally substituteddioxaborolane, dioxaborolanone, dioxaborolandione, dioxaborinane,dioxaborinanone, or dioxaborinandione.

In certain embodiments of any of the foregoing formulae, X is O.

In certain embodiments of any of the foregoing formulae, if R¹ is H,then R³ is not benzyl.

In certain embodiments of any of the foregoing formulae, R¹ is H.

In certain embodiments of any of the foregoing formulae, if R¹ isbenzyl, then R³ is not methyl.

In certain embodiments, R¹ is optionally substituted aralkyl,heteroaralkyl, (cycloalkyl)alkyl, or (heterocycloalkyl)alkyl.

In certain embodiments, R¹ is optionally substituted aralkyl orheteroaralkyl.

In certain such embodiments, R¹ is benzyl.

In other certain such embodiments, R¹ is not benzyl substituted by —CF₃.

In yet other certain such embodiments, R¹ is heteroaralkyl, such as—CH₂-(1H-imidazol-4-yl).

In certain embodiments of any of the foregoing formulae R¹ is optionallysubstituted alkyl, alkenyl, or alkynyl.

In certain such embodiments, R¹ is alkyl, optionally substituted by oneor more substituents independently selected from hydroxy, halo,haloalkyl, alkoxy, —SH, —S-(alkyl), —SeH, —Se-(alkyl), aryl, heteroaryl,cycloalkyl, heterocycloalkyl, amino, carboxylic acid, ester, guanidino,and amido.

In certain such embodiments, R¹ is alkyl, optionally substituted by oneor more substituents independently selected from hydroxy, halo,haloalkyl, alkoxy, —SH, —S-(alkyl), —SeH, —Se-(alkyl), heteroaryl,cycloalkyl, heterocycloalkyl, amino, carboxylic acid, ester, guanidino,and amido.

In certain such embodiments, R¹ is alkyl, optionally substituted by oneor more substituents independently selected from hydroxy, alkoxy,haloalkyl, and —S-(alkyl).

In certain embodiments, R¹ is selected from optionally substitutedcycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

In certain embodiments, R¹ is an amino acid side chain of Arg, His, Lys,Asp, Glu, Ser, Thr, Asn, Gln, Cys, Sec, Gly, Ala, Val, Ile, Leu, Met,Phe, Tyr, or Trp.

In certain embodiments, R¹ and R² are taken together with theintervening atoms to form an optionally substituted 5- to 7-memberedring.

In certain embodiments, R¹ and R² are taken together with theintervening atoms to form an optionally substituted 3- to 7-memberedring, such as a 3-membered ring.

In certain embodiments, R³ is H.

In certain embodiments, R¹ and R³ are taken together with theintervening atoms to form a substituted 5-membered ring.

In certain embodiments, R¹ and R³ are taken together with theintervening atoms to form an optionally substituted 6- or 7-memberedring.

In certain embodiments, R¹ and R³, taken together with the interveningatoms, do not form a tetrahydroisoquinolinyl ring, e.g.,

In certain embodiments, the compound of formula (I) is not:

In certain embodiments, the compound of the invention has a structureselected from:

or a pharmaceutically acceptable salt or prodrug thereof.

In certain embodiments, the compound may be a prodrug, e.g., wherein ahydroxyl in the parent compound is presented as an ester or a carbonate,a carboxylic acid present in the parent compound is presented as anester, or an amino group is presented as an amide. In certain suchembodiments, the prodrug is metabolized to the active parent compound invivo (e.g., the ester is hydrolyzed to the corresponding hydroxyl orcarboxylic acid).

In certain embodiments, the boronic acid may exist in the form of acyclic or linear anhydride. In certain embodiments, the boronic acidexists in the form of a 6-membered ring anhydride, and is also known asa boroxine.

In certain embodiments, arginase inhibitor compounds of the inventionmay be racemic. In certain embodiments, arginase inhibitor compounds ofthe invention may be enriched in one enantiomer. For example, a compoundof the invention may have greater than 30% ee, 40% ee, 50% ee, 60% ee,70% ee, 80% ee, 90% ee, or even 95% or greater ee.

The compounds of the invention have more than one stereocenter.Accordingly, the compounds of the invention may be enriched in one ormore diastereomers. For example, a compound of the invention may havegreater than 30% de, 40% de, 50% de, 60% de, 70% de, 80% de, 90% de, oreven 95% or greater de. In certain embodiments, the compounds of theinvention have substantially one isomeric configuration at one or morestereogenic centers, and have multiple isomeric configurations at theremaining stereogenic centers.

In certain embodiments, the enantiomeric excess of the stereocenterbearing R¹ is at least 40% ee, 50% ee, 60% ee, 70% ee, 80% ee, 90% ee,92% ee, 94% ee, 95% ee, 96% ee, 98% ee or greater ee.

As used herein, single bonds drawn without stereochemistry do notindicate the stereochemistry of the compound. The compound of formula(I) provides an example of a compound for which no stereochemistry isindicated.

As used herein, hashed or bolded non-wedge bonds indicate relative, butnot absolute, stereochemical configuration (e.g., do not distinguishbetween enantiomers of a given diastereomer). For example, in formula(Ia),

the bold, non-wedge bonds indicate that the —CO₂R^(a) group and the(CH₂)₃B(OR^(c))₂ group are configured to be cis to one another, but thebold, non-wedge bonds do not represent the absolute (i.e., R or S)configuration of the compound.

As used herein, hashed or bolded wedge bonds indicate absolutestereochemical configuration. For example, in formula (Ic)

the bold, wedge bond indicates the absolute configuration of thestereocenter to which it is attached, while the bold, non-wedge bondsindicate that the —CO₂R^(a) group and the (CH₂)₃B(OR^(c))₂ group areconfigured to be cis to one another, but do not indicate the absoluteconfiguration of those stereocenters. Therefore, the compound of formula(Ic) represents two isomers in total:

In certain embodiments, a therapeutic preparation of the compound of theinvention may be enriched to provide predominantly one enantiomer of acompound. An enantiomerically enriched mixture may comprise, forexample, at least 60 mol percent of one enantiomer, or more preferablyat least 75, 90, 95, or even 99 mol percent. In certain embodiments, thecompound enriched in one enantiomer is substantially free of the otherenantiomer, wherein substantially free means that the substance inquestion makes up less than 10%, or less than 5%, or less than 4%, orless than 3%, or less than 2%, or less than 1% as compared to the amountof the other enantiomer, e.g., in the composition or compound mixture.For example, if a composition or compound mixture contains 98 grams of afirst enantiomer and 2 grams of a second enantiomer, it would be said tocontain 98 mol percent of the first enantiomer and only 2% of the secondenantiomer.

In certain embodiments, a therapeutic preparation may be enriched toprovide predominantly one diastereomer of the compound of the invention.A diastereomerically enriched mixture may comprise, for example, atleast 60 mol percent of one diastereomer, or more preferably at least75, 90, 95, or even 99 mol percent.

In certain embodiments, the compounds of the invention exhibit animproved pharmacokinetic profile relative to existing arginaseinhibitors.

In certain embodiments, the compounds of the invention exhibit improvedbioavailability relative to existing arginase inhibitors.

Methods of Treatment

Several specific approaches to T-cell activation have shown considerablerecent promise in the treatment of tumors. One such approach involvesactivation of T-cells by blockade of the T-cell surface antigen CTLA-4by the antibody ipilimumab. A second approach is to prevent theactivation of immune checkpoints by blocking the interaction ofprogrammed cell death 1 protein, or PD-1, expressed on T-cells and itsligand, PD-L1 found on many tumors. A third approach is to activate theT-cell receptor by supplying key stimulating factors or nutrients suchas tryptophan.

Inhibitors of indoleamine dioxygenase, or IDO, have been shown torestore extracellular tryptophan without which the T-cell receptorcannot become active. Arginine, like tryptophan, is an amino acid thatis fundamental to the function of cytotoxic T-cells. Without arginine,tumor-specific cytotoxic T-cells fail to express a functional T-cellreceptor on their surface and as a result are unable to activate,proliferate, or mount an effective anti-tumor response. In response totumor-secreted factors, myeloid-derived suppressor cells, or MDSCs,accumulate around the tumor and secrete the enzyme arginase, resultingin depletion of arginine from the tumor microenvironment.

Depletion of arginine due to elevated levels of arginase has beenobserved in renal cell carcinoma and acute myeloid leukemia. Inaddition, significant MDSC infiltrates have been observed in pancreatic,breast and other tumor types. Certain embodiments of the presentinvention provide a method of treating cancer by increasing argininelevels in a tumor microenvironment, thereby allowing activation of thebody's cytotoxic T-cells.

One means of increasing arginine levels in the tumor microenvironment isby inhibiting arginase. Inhibitors of arginase, such as the compounds ofthe invention, may promote an anti-tumor immune response by restoringarginine levels, thereby allowing activation of the body's cytotoxicT-cells.

Accordingly, in certain embodiments, the invention provides methods fortreating or preventing cancer, comprising administering to a subject inneed thereof a therapeutically effective amount of a compound of theinvention (e.g., a compound of Formula I, Ia, Ib, Ic, Id, Ie, If, Ig, orIh), or a pharmaceutical composition comprising said compound.

In certain embodiments, the cancer that is treated by the methods of theinvention is Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia(AML), Adrenocortical Carcinoma, Anal Cancer, Appendix Cancer, AtypicalTeratoid/Rhabdoid Tumor, Basal Cell Carcinoma, Bile Duct Cancer, BladderCancer, Bone Cancer, Brain Tumor, Astrocytoma, Brain and Spinal CordTumor, Brain Stem Glioma, Central Nervous System AtypicalTeratoid/Rhabdoid Tumor, Central Nervous System Embryonal Tumors, BreastCancer, Bronchial Tumors, Burkitt Lymphoma, Carcinoid Tumor, Carcinomaof Unknown Primary, Central Nervous System Cancer, Cervical Cancer,Childhood Cancers, Chordoma, Chronic Lymphocytic Leukemia (CLL), ChronicMyelogenous Leukemia (CML), Chronic Myeloproliferative Disorders, ColonCancer, Colorectal Cancer, Craniopharyngioma, Cutaneous T-Cell Lymphoma,Ductal Carcinoma In Situ (DCIS), Embryonal Tumors, Endometrial Cancer,Ependymoblastoma, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma,Ewing Sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ CellTumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Fibrous Histiocytomaof Bone, Gallbladder Cancer, Gastric Cancer, Gastrointestinal CarcinoidTumor, Gastrointestinal Stromal Tumors (GIST), Germ Cell Tumor,Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Ovarian GermCell Tumor, Gestational Trophoblastic Tumor, Glioma, Hairy CellLeukemia, Head and Neck Cancer, Heart Cancer, Hepatocellular Cancer,Histiocytosis, Langerhans Cell Cancer, Hodgkin Lymphoma, HypopharyngealCancer, Intraocular Melanoma, Islet Cell Tumors, Kaposi Sarcoma, KidneyCancer, Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia, Lipand Oral Cavity Cancer, Liver Cancer, Lobular Carcinoma In Situ (LCIS),Lung Cancer, Lymphoma, AIDS-Related Lymphoma, Macroglobulinemia, MaleBreast Cancer, Medulloblastoma, Medulloepithelioma, Melanoma, MerkelCell Carcinoma, Malignant Mesothelioma, Metastatic Squamous Neck Cancerwith Occult Primary, Midline Tract Carcinoma Involving NUT Gene, MouthCancer, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma/PlasmaCell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndrome,Myelodysplastic/Myeloproliferative Neoplasm, Chronic MyelogenousLeukemia (CML), Acute Myeloid Leukemia (AML), Myeloma, Multiple Myeloma,Chronic Myeloproliferative Disorder, Nasal Cavity Cancer, ParanasalSinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-HodgkinLymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Oral Cavity Cancer,Lip Cancer, Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer,Pancreatic Cancer, Papillomatosis, Paraganglioma, Paranasal SinusCancer, Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer,Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumors ofIntermediate Differentiation, Pineoblastoma, Pituitary Tumor, PlasmaCell Neoplasm, Pleuropulmonary Blastoma, Breast Cancer, Primary CentralNervous System (CNS) Lymphoma, Prostate Cancer, Rectal Cancer, RenalCell Cancer, Renal Pelvis Cancer, Ureter Cancer, Transitional CellCancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,Sarcoma, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, SmallIntestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma, SquamousNeck Cancer with Occult Primary, Stomach Cancer, SupratentorialPrimitive Neuroectodermal Tumors, T-Cell Lymphoma, Testicular Cancer,Throat Cancer, Thymoma, Thymic Carcinoma, Thyroid Cancer, TransitionalCell Cancer of the Renal Pelvis and Ureter, Gestational TrophoblasticTumor, Unknown Primary, Unusual Cancer of Childhood, Urethral Cancer,Uterine Cancer, Uterine Sarcoma, Waldenström Macroglobulinemia, or WilmsTumor.

In certain embodiments, the cancer that is treated by the methods of theinvention is a variety of acute myeloid leukemia (AML), bladder cancer,breast cancer, colorectal cancer, chronic myelogenous leukemia (CML),esophageal cancer, gastric cancer, lung cancer, melanoma, mesothelioma,non-small cell lung carcinoma (NSCLC), ovarian cancer, pancreaticcancer, prostate cancer, renal cancer, or skin cancer.

In certain embodiments, the cancer that is treated by the methods of theinvention is a variety of acute myeloid leukemia (AML), breast cancer,colorectal cancer, chronic myelogenous leukemia (CML), esophagealcancer, gastric cancer, lung cancer, melanoma, non-small cell lungcarcinoma (NSCLC), pancreatic cancer, prostate cancer, or renal cancer.

In certain embodiments, the cancer is selected from bladder cancer,breast cancer (including TNBC), cervical cancer, colorectal cancer,chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma(DLBCL), esophageal adenocarcinoma, glioblastoma, head and neck cancer,leukemia (acute and chronic), low-grade glioma, lung cancer (includingadenocarcinoma, non-small cell lung cancer, and squamous cellcarcinoma), Hodgkin's lymphoma, non-Hodgkin lymphoma (NHL), melanoma,multiple myeloma (MM), ovarian cancer, pancreatic cancer, prostatecancer, renal cancer (including renal clear cell carcinoma and kidneypapillary cell carcinoma), and stomach cancer.

Combination therapy is an important treatment modality in many diseasesettings, such as cancer. Recent scientific advances have increased ourunderstanding of the pathophysiological processes that underlie theseand other complex diseases. This increased understanding has providedimpetus to develop new therapeutic approaches using combinations ofdrugs directed at multiple therapeutic targets to improve treatmentresponse, minimize development of resistance, or minimize adverseevents. In settings in which combination therapy provides significanttherapeutic advantages, there is growing interest in the development ofcombinations with new investigational drugs, such as arginaseinhibitors.

When considering the administration of multiple therapeutic agentstogether, one must be concerned about what sort of drug interactionswill be observed. This action can be positive (when the drug's effect isincreased) or antagonistic (when the drug's effect is decreased) or anew side effect can be produced that neither produces on its own.

When the interaction causes an increase in the effects of one or both ofthe drugs the interaction, the degree to which the final effect of thecombined drugs is greater than administering either drug alone can becalculated resulting in what is called the “combination index” (CI)(Chou and Talalay, 1984). A combination index at or around 1 isconsidered “additive”; whereas a value greater than 1 is considered“synergistic”.

The present invention provides methods for combination therapy intreating or preventing cancer comprising an arginase inhibitor (e.g., acompound of the invention) and one or more additional chemotherapeuticagents.

Certain embodiments of the invention relate to treating cancercomprising conjointly administering a chemotherapeutic agent and acompound of the invention.

In certain embodiments, the chemotherapeutic is an immune-stimulatingagent. For example, the immune-stimulating agent may be apro-inflammatory agent.

The chemotherapeutic agent that may be conjointly administered with thearginase inhibitors described herein in the methods of the inventioninclude aminoglutethimide, amsacrine, anastrozole, asparaginase,AZD5363, Bacillus Calmette-Guérin vaccine (bcg), bicalutamide,bleomycin, bortezomib, buserelin, busulfan, campothecin, capecitabine,carboplatin, carfilzomib, carmustine, chlorambucil, chloroquine,cisplatin, cladribine, clodronate, cobimetinib, colchicine,cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, demethoxyviridin, dexamethasone, dichloroacetate,dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epacadostat,epirubicin, erlotinib, estradiol, estramustine, etoposide, everolimus,exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil,fluoxymesterone, flutamide, gemcitabine, genistein, goserelin,hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan,lenalidomide, letrozole, leucovorin, leuprolide, levamisole, lomustine,lonidamine, mechlorethamine, medroxyprogesterone, megestrol, melphalan,mercaptopurine, mesna, metformin, methotrexate, miltefosine, mitomycin,mitotane, mitoxantrone, MK-2206, nilutamide, nocodazole, octreotide,olaparib, oxaliplatin, paclitaxel, pamidronate, pazopanib, pentostatin,perifosine, plicamycin, pomalidomide, porfimer, procarbazine,raltitrexed, rituximab, rucaparib, selumetinib, sorafenib, streptozocin,sunitinib, suramin, talazoparib, tamoxifen, temozolomide, temsirolimus,teniposide, testosterone, thalidomide, thioguanine, thiotepa, titanocenedichloride, topotecan, trametinib, trastuzumab, tretinoin, veliparib,vinblastine, vincristine, vindesine, or vinorelbine.

In certain embodiments, the chemotherapeutic agent that may beadministered with the arginase inhibitors described herein in themethods of the invention include abagovomab, adecatumumab, afutuzumab,anatumomab mafenatox, apolizumab, atezolizumab, blinatumomab,catumaxomab, durvalumab, epacadostat, epratuzumab, inotuzumabozogamicin, intelumumab, ipilimumab, isatuximab, lambrolizumab,nivolumab, ocaratuzumab, olatatumab, pembrolizumab, pidilizumab,ticilimumab, samalizumab, or tremelimumab.

In certain embodiments, the chemotherapeutic agent is ipilimumab,nivolumab, pembrolizumab, or pidilizumab.

Many combination therapies have been developed for the treatment ofcancer. In certain embodiments, compounds of the invention may beconjointly administered with a combination therapy. Examples ofcombination therapies with which compounds of the invention may beconjointly administered are included in Table 1.

TABLE 1 Exemplary combinatorial therapies for the treatment of cancer.Name Therapeutic agents ABV Doxorubicin, Bleomycin, Vinblastine ABVDDoxorubicin, Bleomycin, Vinblastine, Dacarbazine AC (Breast)Doxorubicin, Cyclophosphamide AC (Sarcoma) Doxorubicin, Cisplatin AC(Neuroblastoma) Cyclophosphamide, Doxorubicin ACE Cyclophosphamide,Doxorubicin, Etoposide ACe Cyclophosphamide, Doxorubicin AD Doxorubicin,Dacarbazine AP Doxorubicin, Cisplatin ARAC-DNR Cytarabine, DaunorubicinB-CAVe Bleomycin, Lomustine, Doxorubicin, Vinblastine BCVPP Carmustine,Cyclophosphamide, Vinblastine, Procarbazine, Prednisone BEACOPPBleomycin, Etoposide, Doxorubicin, Cyclophosphamide, Vincristine,Procarbazine, Prednisone, Filgrastim BEP Bleomycin, Etoposide, CisplatinBIP Bleomycin, Cisplatin, Ifosfamide, Mesna BOMP Bleomycin, Vincristine,Cisplatin, Mitomycin CA Cytarabine, Asparaginase CABO Cisplatin,Methotrexate, Bleomycin, Vincristine CAF Cyclophosphamide, Doxorubicin,Fluorouracil CAL-G Cyclophosphamide, Daunorubicin, Vincristine,Prednisone, Asparaginase CAMP Cyclophosphamide, Doxorubicin,Methotrexate, Procarbazine CAP Cyclophosphamide, Doxorubicin, CisplatinCaT Carboplatin, Paclitaxel CAV Cyclophosphamide, Doxorubicin,Vincristine CAVE ADD CAV and Etoposide CA-VP16 Cyclophosphamide,Doxorubicin, Etoposide CC Cyclophosphamide, Carboplatin CDDP/VP-16Cisplatin, Etoposide CEF Cyclophosphamide, Epirubicin, FluorouracilCEPP(B) Cyclophosphamide, Etoposide, Prednisone, with or without/Bleomycin CEV Cyclophosphamide, Etoposide, Vincristine CF Cisplatin,Fluorouracil or Carboplatin Fluorouracil CHAP Cyclophosphamide orCyclophosphamide, Altretamine, Doxorubicin, Cisplatin ChlVPPChlorambucil, Vinblastine, Procarbazine, Prednisone CHOPCyclophosphamide, Doxorubicin, Vincristine, Prednisone CHOP-BLEO AddBleomycin to CHOP CISCA Cyclophosphamide, Doxorubicin, CisplatinCLD-BOMP Bleomycin, Cisplatin, Vincristine, Mitomycin CMF Methotrexate,Fluorouracil, Cyclophosphamide CMFP Cyclophosphamide, Methotrexate,Fluorouracil, Prednisone CMFVP Cyclophosphamide, Methotrexate,Fluorouracil, Vincristine, Prednisone CMV Cisplatin, Methotrexate,Vinblastine CNF Cyclophosphamide, Mitoxantrone, Fluorouracil CNOPCyclophosphamide, Mitoxantrone, Vincristine, Prednisone COB Cisplatin,Vincristine, Bleomycin CODE Cisplatin, Vincristine, Doxorubicin,Etoposide COMLA Cyclophosphamide, Vincristine, Methotrexate, Leucovorin,Cytarabine COMP Cyclophosphamide, Vincristine, Methotrexate, PrednisoneCooper Regimen Cyclophosphamide, Methotrexate, Fluorouracil,Vincristine, Prednisone COP Cyclophosphamide, Vincristine, PrednisoneCOPE Cyclophosphamide, Vincristine, Cisplatin, Etoposide COPPCyclophosphamide, Vincristine, Procarbazine, Prednisone CP(ChronicChlorambucil, Prednisone lymphocytic leukemia) CP (Ovarian Cancer)Cyclophosphamide, Cisplatin CT Cisplatin, Paclitaxel CVD Cisplatin,Vinblastine, Dacarbazine CVI Carboplatin, Etoposide, Ifosfamide, MesnaCVP Cyclophosphamide Vincristine, Prednisome CVPP Lomustine,Procarbazine, Prednisone CYVADIC Cyclophosphamide, Vincristine,Doxorubicin, Dacarbazine DA Daunorubicin, Cytarabine DAT Daunorubicin,Cytarabine, Thioguanine DAV Daunorubicin, Cytarabine, Etoposide DCTDaunorubicin, Cytarabine, Thioguanine DHAP Cisplatin, Cytarabine,Dexamethasone DI Doxorubicin, Ifosfamide DTIC/Tamoxifen Dacarbazine,Tamoxifen DVP Daunorubicin, Vincristine, Prednisone EAP Etoposide,Doxorubicin, Cisplatin EC Etoposide, Carboplatin EFP Etoposie,Fluorouracil, Cisplatin ELF Etoposide, Leucovorin, Fluorouracil EMA 86Mitoxantrone, Etoposide, Cytarabine EP Etoposide, Cisplatin EVAEtoposide, Vinblastine FAC Fluorouracil, Doxorubicin, CyclophosphamideFAM Fluorouracil, Doxorubicin, Mitomycin FAMTX Methotrexate, Leucovorin,Doxorubicin FAP Fluorouracil, Doxorubicin, Cisplatin F-CL Fluorouracil,Leucovorin FEC Fluorouracil, Cyclophosphamide, Epirubicin FEDFluorouracil, Etoposide, Cisplatin FL Flutamide, Leuprolide FZFlutamide, Goserelin acetate implant HDMTX Methotrexate, LeucovorinHexa-CAF Altretamine, Cyclophosphamide, Methotrexate, Fluorouracil ICE-TIfosfamide, Carboplatin, Etoposide, Paclitaxel, Mesna IDMTX/6-MPMethotrexate, Mercaptopurine, Leucovorin IE Ifosfamide, Etoposie, MesnaIfoVP Ifosfamide, Etoposide, Mesna IPA Ifosfamide, Cisplatin,Doxorubicin M-2 Vincristine, Carmustine, Cyclophosphamide, Prednisone,Melphalan MAC-III Methotrexate, Leucovorin, Dactinomycin,Cyclophosphamide MACC Methotrexate, Doxorubicin, Cyclophosphamide,Lomustine MACOP-B Methotrexate, Leucovorin, Doxorubicin,Cyclophosphamide, Vincristine, Bleomycin, Prednisone MAID Mesna,Doxorubicin, Ifosfamide, Dacarbazine m-BACOD Bleomycin, Doxorubicin,Cyclophosphamide, Vincristine, Dexamethasone, Methotrexate, LeucovorinMBC Methotrexate, Bleomycin, Cisplatin MC Mitoxantrone, Cytarabine MFMethotrexate, Fluorouracil, Leucovorin MICE Ifosfamide, Carboplatin,Etoposide, Mesna MINE Mesna, Ifosfamide, Mitoxantrone, Etoposidemini-BEAM Carmustine, Etoposide, Cytarabine, Melphalan MOBP Bleomycin,Vincristine, Cisplatin, Mitomycin MOP Mechlorethamine, Vincristine,Procarbazine MOPP Mechlorethamine, Vincristine, Procarbazine, PrednisoneMOPP/ABV Mechlorethamine, Vincristine, Procarbazine, Prednisone,Doxorubicin, Bleomycin, Vinblastine MP (multiple Melphalan, Prednisonemyeloma) MP (prostate cancer) Mitoxantrone, Prednisone MTX/6-MOMethotrexate, Mercaptopurine MTX/6-MP/VP Methotrexate, Mercaptopurine,Vincristine, Prednisone MTX-CDDPAdr Methotrexate, Leucovorin, Cisplatin,Doxorubicin MV (breast cancer) Mitomycin, Vinblastine MV (acutemyelocytic Mitoxantrone, Etoposide leukemia) M-VAC MethotrexateVinblastine, Doxorubicin, Cisplatin MVP Mitomycin Vinblastine, CisplatinMVPP Mechlorethamine, Vinblastine, Procarbazine, Prednisone NFLMitoxantrone, Fluorouracil, Leucovorin NOVP Mitoxantrone, Vinblastine,Vincristine OPA Vincristine, Prednisone, Doxorubicin OPPA AddProcarbazine to OPA. PAC Cisplatin, Doxorubicin PAC-I Cisplatin,Doxorubicin, Cyclophosphamide PA-CI Cisplatin, Doxorubicin PCPaclitaxel, Carboplatin or Paclitaxel, Cisplatin PCV Lomustine,Procarbazine, Vincristine PE Paclitaxel, Estramustine PFL Cisplatin,Fluorouracil, Leucovorin POC Prednisone, Vincristine, Lomustine ProMACEPrednisone, Methotrexate, Leucovorin, Doxorubicin, Cyclophosphamide,Etoposide ProMACE/cytaBOM Prednisone, Doxorubicin, Cyclophosphamide,Etoposide, Cytarabine, Bleomycin, Vincristine, Methotrexate, Leucovorin,Cotrimoxazole PRoMACE/MOPP Prednisone, Doxorubicin, Cyclophosphamide,Etoposide, Mechlorethamine, Vincristine, Procarbazine, Methotrexate,Leucovorin Pt/VM Cisplatin, Teniposide PVA Prednisone, Vincristine,Asparaginase PVB Cisplatin, Vinblastine, Bleomycin PVDA Prednisone,Vincristine, Daunorubicin, Asparaginase SMF Streptozocin, Mitomycin,Fluorouracil TAD Mechlorethamine, Doxorubicin, Vinblastine, Vincristine,Bleomycin, Etoposide, Prednisone TCF Paclitaxel, Cisplatin, FluorouracilTIP Paclitaxel, Ifosfamide, Mesna, Cisplatin TTT Methotrexate,Cytarabine, Hydrocortisone Topo/CTX Cyclophosphamide, Topotecan, MesnaVAB-6 Cyclophosphamide, Dactinomycin, Vinblastine, Cisplatin, BleomycinVAC Vincristine, Dactinomycin, Cyclophosphamide VACAdr Vincristine,Cyclophosphamide, Doxorubicin, Dactinomycin, Vincristine VADVincristine, Doxorubicin, Dexamethasone VATH Vinblastine, Doxorubicin,Thiotepa, Flouxymesterone VBAP Vincristine, Carmustine, Doxorubicin,Prednisone VBCMP Vincristine, Carmustine, Melphalan, Cyclophosphamide,Prednisone VC Vinorelbine, Cisplatin VCAP Vincristine, Cyclophosphamide,Doxorubicin, Prednisone VD Vinorelbine, Doxorubicin VelP Vinblastine,Cisplatin, Ifosfamide, Mesna VIP Etoposide, Cisplatin, Ifosfamide, MesnaVM Mitomycin, Vinblastine VMCP Vincristine, Melphalan, Cyclophosphamide,Prednisone VP Etoposide, Cisplatin V-TAD Etoposide, Thioguanine,Daunorubicin, Cytarabine 5 + 2 Cytarabine, Daunorubicin, Mitoxantrone7 + 3 Cytarabine with/, Daunorubicin or Idarubicin or Mitoxantrone “8 in1” Methylprednisolone, Vincristine, Lomustine, Procarbazine,Hydroxyurea, Cisplatin, Cytarabine, Dacarbazine

In certain embodiments, the conjointly administered chemotherapeuticagent is selected from a metabolic enzyme inhibitor, such as glucosetransporters, hexokinase, pyruvate kinase M2, lactate dehydrogenase 1 or2, pyruvate dehydrogenase kinase, fatty acid synthase and glutaminase.In some embodiments, the inhibitor inhibits lactate dehydrogenase 1 or2, or glutaminase. In certain embodiments, the inhibitor is CB-839.

Immune-targeted agents (also known as immuno-oncology agents) actagainst tumors by modulating immune cells. The field of cancerimmunotherapy is rapidly growing, with new targets constantly beingidentified (Chen and Mellman, 2013; Morrissey et al., 2016; Kohrt etal., 2016). The present invention provides a combination of animmuno-oncology agent and a glutaminase inhibitor.

Examples of immuno-oncology agents comprise agents that modulate immunecheckpoints such as 2B4, 4-1BB (CD137), AaR, B7-H3, B7-H4, BAFFR, BTLA,CD2, CD7, CD27, CD28, CD30, CD40, CD80, CD83 ligand, CD86, CD160, CD200,CDS, CEACAM, CTLA-4, GITR, HVEM, ICAM-1, KIR, LAG-3, LAIR1, LFA-1(CD11a/CD18), LIGHT, NKG2C, NKp80, OX40, PD-1, PD-L1, PD-L2, SLAMF7,TGFRβ, TIGIT, Tim3 and VISTA.

Immuno-oncology agents may be in the form of antibodies, peptides, smallmolecules or viruses.

In some embodiments, the conjointly administered chemotherapeutic agentis an immuno-oncology therapeutic agent, such as an inhibitor ofarginase, CTLA-4, indoleamine 2,3-dioxygenase, and/or PD-1/PD-L1. Incertain embodiments, the immuno-oncology therapeutic agent isabagovomab, adecatumumab, afutuzumab, alemtuzumab, anatumomab mafenatox,apolizumab, atezolizumab, avelumab, blinatumomab, BMS-936559,catumaxomab, durvalumab, epacadostat, epratuzumab, indoximod, inotuzumabozogamicin, intelumumab, ipilimumab, isatuximab, lambrolizumab,MED14736, MPDL3280A, nivolumab, ocaratuzumab, ofatumumab, olatatumab,pembrolizumab, pidilizumab, rituximab, ticilimumab, samalizumab, ortremelimumab. Alternatively, the immuno-oncology therapeutic agent isabagovomab, adecatumumab, afutuzumab, anatumomab mafenatox, apolizumab,atezolizumab, blinatumomab, catumaxomab, durvalumab, epacadostat,epratuzumab, indoximod, inotuzumab ozogamicin, intelumumab, ipilimumab,isatuximab, lambrolizumab, nivolumab, ocaratuzumab, olatatumab,pembrolizumab, pidilizumab, ticilimumab, samalizumab, or tremelimumab.In some embodiments, the immuno-oncology agent is indoximod, ipilimumab,nivolumab, pembrolizumab, or pidilizumab. In certain embodiments, theimmuno-oncology therapeutic agent is ipilimumab.

Exemplary immuno-oncology agents are disclosed in Adams, J. L. et al.“Big Opportunities for Small Molecules in Immuno-Oncology” NatureReviews Drug Discovery 2015, 14, page 603-621, the contents of which arehereby incorporated by reference.

In certain embodiments, the conjointly administered chemotherapeuticagent is a pro-inflammatory agent. In certain embodiments, thepro-inflammatory agent administered with the arginase inhibitors of theinvention is a cytokine or a chemokine.

Pro-inflammatory cytokines are produced predominantly by activatedmacrophages and are involved in the up-regulation of inflammatoryreactions.

Exemplary pro-inflammatory cytokines include IL-1, IL-1β, IL-6, IL-8,TNF-α, and IFN-γ.

Chemokines are a group of small cytokines. Pro-inflammatory chemokinespromote recruitment and activation of multiple lineages of leukocytes(e.g., lymphocytes, macrophages). Chemokines are related in primarystructure and share several conserved amino acid residues. Inparticular, chemokines typically include two or four cysteine residuesthat contribute to the three-dimensional structure via formation ofdisulfide bonds. Chemokines may be classified in one of four groups: C-Cchemokines, C-X-C chemokines, C chemokines, and C-X₃-C chemokines. C-X-Cchemokines include a number of potent chemoattractants and activators ofneutrophils, such as interleukin 8 (IL-8), PF4 and neutrophil-activatingpeptide-2 (NAP-2). The C-C chemokines include, for example, RANTES(Regulated on Activation, Normal T Expressed and Secreted), macrophageinflammatory proteins 1-alpha and 1-beta (MIP-1α and MIP-1β), eotaxinand human monocyte chemotactic proteins 1 to 3 (MCP-1, MCP-2, MCP-3),which have been characterized as chemoattractants and activators ofmonocytes or lymphocytes. Accordingly, exemplary pro-inflammatorychemokines include MIP-1α, MIP-1β, MIP-17, MCP-1, MCP-2, MCP-3, IL-8,PF4, NAP-2, RANTES, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL2, CXCL8, andCXCL10.

In certain embodiments, the method of treating or preventing cancerfurther comprises administering one or more non-chemical methods ofcancer treatment, such as radiation therapy, surgery, thermoablation,focused ultrasound therapy, cryotherapy, or a combination of theforegoing.

Cellular pathways operate more like webs than superhighways. There aremultiple redundancies, or alternate routes, that are activated inresponse to the inhibition of a pathway. This redundancy promotes theemergence of resistant cells or organisms under the selective pressureof a targeted agent, resulting in drug resistance and clinical relapse.

In certain embodiments of the invention, the chemotherapeutic agent isadministered simultaneously with the arginase inhibitor. In certainembodiments, the chemotherapeutic agent is administered within about 5minutes to within about 168 hours prior or after of the arginaseinhibitor.

The present invention provides combination therapies comprising animmuno-oncology agent selected from inhibitors of CTLA-4, indoleamine2,3-dioxygenase, and PD-1/PD-L1, and an arginase inhibitor of formula(I). In certain embodiments, the combination therapy treats or preventscancer, an immunological disorder, or a chronic infection.

In certain embodiments, the invention provides methods for treating orpreventing an immunological disease, comprising administering to asubject in need thereof a therapeutically effective amount of a compoundof the invention (e.g., a compound of Formula I, Ia, Ib, Ic, Id, Ie, If,Ig, or Ih), or a pharmaceutical composition comprising said compound.

In certain embodiments, the immunological disease is selected fromankylosing spondylitis, Crohn's disease, erythema nodosum leprosum(ENL), graft versus host disease (GVHD), HIV-associated wastingsyndrome, lupus erythematosus, organ transplant rejection,post-polycythemia, psoriasis, psoriatic arthritis, recurrent aphthousulcers, rheumatoid arthritis (RA), severe recurrent aphthous stomatitis,systemic sclerosis, and tuberous sclerosis.

In certain embodiments, the method for treating or preventing animmunological disease further comprises conjointly administering animmuno-oncology therapeutic agent, as described above.

In certain embodiments, the invention provides methods for treating orpreventing a chronic infection, comprising administering to a subject inneed thereof a therapeutically effective amount of a compound of theinvention (e.g., a compound of Formula I, Ia, Ib, Ic, Id, Ie, If, Ig, orIh), or a pharmaceutical composition comprising said compound.

In certain embodiments, the chronic infection is selected from bladderinfection, chronic fatigue syndrome, cytomegalovirus/epstein barr virus,fibromyalgia, hepatitis B virus (HBV), hepatitis C virus (HCV), HIV/AIDSvirus, mycoplasma infection, and urinary tract infections.

In certain embodiments, the method for treating or preventing a chronicinfection further comprises conjointly administering an immuno-oncologytherapeutic agent, as described above.

Arginase plays multiple major roles within the body. In addition tomodulating immune responses, arginase is involved in regulating nitricoxide levels effecting vasodilation and bronchodilation (Jung et al.,2010; Morris, 2010). Fibrosis and remodeling also relies on arginaseactivity that functions in upstream processes for proline, collagen andpolyamine production (Kitowska et al., 2008; Grasemann et al., 2015).

In certain embodiments, the invention provides a method for thetreatment or prevention of a disease or condition associated withexpression or activity of arginase I, arginase II, or a combinationthereof in a subject, comprising administering to the subject atherapeutically effective amount of at least one of formula (I), or apharmaceutically acceptable salt or stereoisomer thereof.

In certain embodiments, the disease or condition is selected fromcardiovascular disorders, sexual disorders, wound healing disorders,gastrointestinal disorders, autoimmune disorders, immune disorders,infections, pulmonary disorders and hemolytic disorders.

In certain embodiments, the disease or condition is a cardiovasculardisorder selected from systemic hypertension, interstitial lung disease,pulmonary arterial hypertension (PAH), pulmonary arterial hypertensionin high altitude, ischemia reperfusion (IR) injury, myocardialinfarction, and atherosclerosis.

In certain embodiments, the disease or condition is pulmonary arterialhypertension (PAH).

In certain embodiments, the disease or condition is myocardialinfarction or atherosclerosis.

In certain embodiments, the disease or condition is a pulmonary disorderselected from chemically-induced lung fibrosis, idiopathic pulmonaryfibrosis, cystic fibrosis, chronic obstructive pulmonary disease (COPD),and asthma.

In certain embodiments, the disease or condition is an autoimmunedisorder selected from encephalomyelitis, multiple sclerosis,anti-phospholipid syndrome 1, autoimmune hemolytic anaemia, chronicinflammatory demyelinating polyradiculoneuropathy, dermatitisherpetiformis, dermatomyositis, myasthenia gravis, pemphigus, rheumatoidarthritis, stiff-person syndrome, type 1 diabetes, ankylosingspondylitis, paroxysmal nocturnal hemoglobinuria (PNH), paroxysmal coldhemoglobinuria, severe idiopathic autoimmune hemolytic anemia, andGoodpasture's syndrome.

In certain embodiments, the disease or condition is an immune disorderselected from myeloid-derived suppressor cell (MDSC) mediated T-celldysfunction, human immunodeficiency virus (HIV), autoimmuneencephalomyelitis, and ABO mismatch transfusion reaction.

In certain embodiments, the disease or condition is myeloid-derivedsuppressor cell (MDSC) mediated T-cell dysfunction.

In certain embodiments, the disease or condition is a hemolytic disorderselected from sickle-cell disease, thalassemias, hereditaryspherocytosis, stomatocytosis, microangiopathic hemolytic anemiaspyruvate kinase deficiency, infection-induced anemia, cardiopulmonarybypass and mechanical heart valve-induced anemia, and chemical inducedanemia.

In certain embodiments, the disease or condition is a gastrointestinaldisorder selected from gastrointestinal motility disorders, gastriccancer, inflammatory bowel disease, Crohn's disease, ulcerative colitis,and gastric ulcer.

In certain embodiments, the disease or condition is a sexual disorderselected from Peyronie's Disease and erectile dysfunction.

In certain embodiments, the disease or condition is an infectionselected from a parasitic infection, a viral infection, and a bacterialinfection. In certain ebodiments the bacterial infection istuberculosis.

In certain embodiments, the disease or condition is ischemia reperfusion(IR) injury selected from liver IR, kidney IR, and myocardial IR.

In certain embodiments, the disease or condition is selected from renaldisease inflammation, psoriasis, leishmaniasis, neurodegenerativediseases, wound healing, human immunodeficiency virus (HIV), hepatitis Bvirus (HBV), H. pylori infections, fibrotic disorders, arthritis,candidiasis, periodontal disease, keloids, adenotonsillar disease,African sleeping sickness and Chagas' disease.

In certain embodiments, the disease or condition is a wound healingdisorder selected from infected and uninfected wound healing.

In further embodiments, the present invention provides a method ofidentifying a therapeutic agent effective to increase the level ofarginine in a tumor, comprising:

a) measuring a first level of arginine in a tumor;b) contacting the tumor with a therapeutic agent, such as a compound offormula (I);andc) measuring a second level of arginine in the tumor;

wherein when the second level of arginine is higher than the first levelof arginine, then the therapeutic agent is effective to increase thelevel of arginine in the tumor.

In certain embodiments, this method is conducted in vitro. Inalternative embodiments, this method is conducted in vivo.

In certain embodiments (e.g., when the method is conducted in vivo), thestep of contacting the tumor with a therapeutic agent comprisingadministering the therapeutic agent to a subject. In certainembodiments, the subject can be a human.

A level of arginine may be measured, for example, by HPLC, massspectrometry, LCMS, or other analytic techiques known to those of skillin the art. The invention also provides a method of identifying atherapeutic agent effective to increase the level of arginine in a tumorin a subject, comprising:

a) measuring a first level of arginine in a tumor of a subject;b) administering to the subject a therapeutic agent, such as a compoundof formula (I); andc) measuring a second level of arginine in the tumor of the subject;

wherein when the second level of arginine is higher than the first levelof arginine, then the therapeutic agent is effective to increase thelevel of arginine in the tumor of the subject.

In certain embodiments, the step of administering comprises oraladministration of the therapeutic agent. Alternatively, the step ofadministering can comprise parenteral administration of the therapeuticagent. Further methods of administration are discussed herein.

In certain embodiments, the subject is a human.

As used herein, the term “in a tumor” refers to the entire tumor massand the tumor microenvironment. For example, the tumor mass can include,but is in no way limited to, cancer (tumorous) cells, T-cells,macrophages, and stromal cells. The “tumor microenvironment” is anart-recognized term and refers to the cellular environment in which thetumor exists, and includes, for example, surrounding blood vessels,immune cells, other cells, fibroblasts, signaling molecules, and theextracellular matrix. Therefore, measurement of arginine “in a tumor”refers to measurement of arginine in the tumor mass or in itsmicroenvironment.

Accordingly, in certain embodiments of the methods described herein, thefirst and second levels of arginine are measured in the tumor cells.

In other embodiments, the first and second levels of arginine aremeasured in stromal cells associated with the tumor.

In certain embodiments, the therapeutic agent is a compound of Formula(I). Exemplary compounds are described herein.

In certain embodiments in which the therapeutic agent is effective toincrease the level of arginine in a tumor, the therapeutic agent can beeffective to treat the tumor.

In other embodiments, the present invention provides a method ofassessing a response of a tumor to an agent of arginine therapy,comprising:

a) measuring a first level of arginine in a tumor of a cancer patient;b) administering to the patient an agent of arginine therapy; andc) measuring a second level of arginine in the tumor of the patient,thereby assessing the response of the tumor to the agent of argininetherapy.

In certain embodiments, if the second level of arginine is higher thanthe first level of arginine, then the tumor is responsive to (i.e., istreated by) the agent of arginine therapy. An increase of arginine in atumor mass or in the tumor microenvironment can indicate an increase inthe number of cytotoxic T-cells or an increase in the activity ofcytotoxic T-cells.

An “agent of arginine therapy” as used herein, means a therapeutic agentthat can cause an increase in the level of arginine in the system ofinterest (e.g., a tumor mass and its microenvironment). Preferably, theagent of arginine therapy is an arginase inhibitor. More preferably, thearginase inhibitor is a compound of Formula (I).

In other embodiments, the present invention provides a method ofassessing the anti-cancer efficacy of an agent of arginine therapy,comprising:

a) measuring a first level of arginine in a tumor of a cancer patient;b) administering to the patient an agent of arginine therapy; andc) measuring a second level of arginine in the tumor of the patient,thereby assessing the anti-cancer efficacy of an agent of argininetherapy.

In certain embodiments, when the second level of arginine is higher thanthe first level of arginine, then the agent of arginine therapy isefficacious for treating cancer in a patient.

In certain embodiments, the agent of arginine therapy is an arginaseinhibitor.

The present invention also provides a method for treating or preventingcancer, comprising conjointly administering to a subject in need thereofa therapeutically effective amount of an agent of arginine therapy andone or more additional chemotherapeutic agents.

In certain embodiments, administering the agent of arginine therapyeffects an increase in a level of arginine in a tumor of the subjectrelative to the level of arginine in the tumor prior to administration.

In certain embodiments, administering the agent of arginine therapyeffects an increase in a level of arginine in the tumor cells of thesubject relative to the level of arginine in the tumor cells prior toadministration.

Similarly, administering the agent of arginine therapy may effect anincrease in a level of arginine in stromal cells associated with thetumor of the subject relative to the level of arginine in the stromalcells prior to administration.

In certain embodiments, the agent of arginine therapy is an arginaseinhibitor. A number of exemplary arginase inhibitors are describedherein. In particular embodiments, the arginase inhibitor is a compoundhaving the structure of any one of Formulae I, Ia, Ib, Ic, Id, Ie, If,Ig, or Ih, which are described herein.

In other embodiments, the invention provides methods for assessing theanti-cancer efficacy of a combination therapy regimen, comprising:

a) measuring a first level of arginine in a tumor of a cancer patient;b) conjointly administering to the patient an agent of arginine therapyand one or more additional chemotherapeutic agents; andc) measuring a second level of arginine in the tumor of the patient,thereby assessing the anti-cancer efficacy of the combination therapyregimen.

In certain embodiments, when the second level of arginine is higher thanthe first level of arginine, then the combination therapy regimen isefficacious for treating cancer in the patient.

In certain embodiments, the agent of arginine therapy used in thecombination therapy regimen is an arginase inhibitor, such as a compoundof any one of Formulae I, Ia, Ib, Ic, Id, Ie, If, Ig, or Ih.

In certain embodiments, the combination therapy regimen is moreefficacious than a therapy regimen of the arginase inhibitor as a singleagent, or a therapy regimen of the additional chemotherapeutic agent asa single agent.

Definitions

The term “acyl” is art-recognized and refers to a group represented bythe general formula hydrocarbylC(O)—, preferably alkylC(O)—.

The term “acylamino” is art-recognized and refers to an amino groupsubstituted with an acyl group and may be represented, for example, bythe formula hydrocarbylC(O)NH—.

The term “acyloxy” is art-recognized and refers to a group representedby the general formula hydrocarbylC(O)O—, preferably alkylC(O)O—.

The term “alkoxy” refers to an alkyl group, preferably a lower alkylgroup, having an oxygen attached thereto. Representative alkoxy groupsinclude methoxy, ethoxy, propoxy, tert-butoxy and the like.

The term “alkoxyalkyl” refers to an alkyl group substituted with analkoxy group and may be represented by the general formulaalkyl-O-alkyl.

The term “alkenyl”, as used herein, refers to an aliphatic groupcontaining at least one double bond and is intended to include both“unsubstituted alkenyls” and “substituted alkenyls”, the latter of whichrefers to alkenyl moieties having substituents replacing a hydrogen onone or more carbons of the alkenyl group. Such substituents may occur onone or more carbons that are included or not included in one or moredouble bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed below, except where stability isprohibitive. For example, substitution of alkenyl groups by one or morealkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated.

An “alkyl” group or “alkane” is a straight chained or branchednon-aromatic hydrocarbon which is completely saturated. Typically, astraight chained or branched alkyl group has from 1 to about 20 carbonatoms, preferably from 1 to about 10 unless otherwise defined. Examplesof straight chained and branched alkyl groups include methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl,pentyl and octyl. A C₁-C₆ straight chained or branched alkyl group isalso referred to as a “lower alkyl” group.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing one or morehydrogens on one or more carbons of the hydrocarbon backbone. Suchsubstituents, if not otherwise specified, can include, for example, ahalogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl,a formyl, or an acyl), a thiocarbonyl (such as a thioester, athioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, aphosphonate, a phosphinate, an amino, an amido, an amidine, an imine, acyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, asulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, anaralkyl, a guanidino, or an aromatic or heteroaromatic moiety. It willbe understood by those skilled in the art that the moieties substitutedon the hydrocarbon chain can themselves be substituted, if appropriate.For instance, the substituents of a substituted alkyl may includesubstituted and unsubstituted forms of amino, azido, imino, amido,phosphoryl (including phosphonate and phosphinate), sulfonyl (includingsulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, aswell as ethers, alkylthios, carbonyls (including ketones, aldehydes,carboxylates, and esters), —CF₃, —CN and the like. Exemplary substitutedalkyls are described below. Cycloalkyls can be further substituted withalkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substitutedalkyls, —CF₃, —CN, and the like.

The term “C_(x-y)” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups that contain from x to y carbons in the chain. Forexample, the term “C_(x-y)alkyl” refers to substituted or unsubstitutedsaturated hydrocarbon groups, including straight-chain alkyl andbranched-chain alkyl groups that contain from x to y carbons in thechain, including haloalkyl groups such as trifluoromethyl and2,2,2-trifluoroethyl, etc. C₀ alkyl indicates a hydrogen where the groupis in a terminal position, a bond if internal. The terms“C_(2-y)alkenyl” and “C_(2-y)alkynyl” refer to substituted orunsubstituted unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double or triple bond respectively.

The term “alkylamino”, as used herein, refers to an amino groupsubstituted with at least one alkyl group.

The term “alkylthio”, as used herein, refers to a thiol groupsubstituted with an alkyl group and may be represented by the generalformula alkylS—.

The term “alkynyl”, as used herein, refers to an aliphatic groupcontaining at least one triple bond and is intended to include both“unsubstituted alkynyls” and “substituted alkynyls”, the latter of whichrefers to alkynyl moieties having substituents replacing a hydrogen onone or more carbons of the alkynyl group. Such substituents may occur onone or more carbons that are included or not included in one or moretriple bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed above, except where stability isprohibitive. For example, substitution of alkynyl groups by one or morealkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated.

The term “amide”, as used herein, refers to a group

wherein each R¹⁰ independently represent a hydrogen or hydrocarbylgroup, or two R¹⁰ are taken together with the N atom to which they areattached complete a heterocycle having from 4 to 8 atoms in the ringstructure.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by

wherein each R¹⁰ independently represents a hydrogen or a hydrocarbylgroup, or two R¹⁰ are taken together with the N atom to which they areattached complete a heterocycle having from 4 to 8 atoms in the ringstructure.

The term “aminoalkyl”, as used herein, refers to an alkyl groupsubstituted with an amino group.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group.

The term “aryl” as used herein include substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 5- to 7-membered ring, more preferably a6-membered ring. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings is aromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groupsinclude benzene, naphthalene, phenanthrene, phenol, aniline, and thelike.

The term “carbamate” is art-recognized and refers to a group

wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbylgroup, such as an alkyl group, or R⁹ and R¹⁰ taken together with theintervening atom(s) complete a heterocycle having from 4 to 8 atoms inthe ring structure.

The terms “carbocycle”, and “carbocyclic”, as used herein, refers to asaturated or unsaturated ring in which each atom of the ring is carbon.The term carbocycle includes both aromatic carbocycles and non-aromaticcarbocycles. Non-aromatic carbocycles include both cycloalkane rings, inwhich all carbon atoms are saturated, and cycloalkene rings, whichcontain at least one double bond. “Carbocycle” includes 5-7 memberedmonocyclic and 8-12 membered bicyclic rings. Each ring of a bicycliccarbocycle may be selected from saturated, unsaturated and aromaticrings. Carbocycle includes bicyclic molecules in which one, two or threeor more atoms are shared between the two rings. The term “fusedcarbocycle” refers to a bicyclic carbocycle in which each of the ringsshares two adjacent atoms with the other ring. Each ring of a fusedcarbocycle may be selected from saturated, unsaturated and aromaticrings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, maybe fused to a saturated or unsaturated ring, e.g., cyclohexane,cyclopentane, or cyclohexene. Any combination of saturated, unsaturatedand aromatic bicyclic rings, as valence permits, is included in thedefinition of carbocyclic. Exemplary “carbocycles” include cyclopentane,cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene,1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene andadamantane. Exemplary fused carbocycles include decalin, naphthalene,1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane,4,5,6,7-tetrahydro-1H-indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles”may be substituted at any one or more positions capable of bearing ahydrogen atom.

A “cycloalkyl” group is a cyclic hydrocarbon which is completelysaturated. “Cycloalkyl” includes monocyclic and bicyclic rings.Typically, a monocyclic cycloalkyl group has from 3 to about 10 carbonatoms, more typically 3 to 8 carbon atoms unless otherwise defined. Thesecond ring of a bicyclic cycloalkyl may be selected from saturated,unsaturated and aromatic rings. Cycloalkyl includes bicyclic moleculesin which one, two or three or more atoms are shared between the tworings. The term “fused cycloalkyl” refers to a bicyclic cycloalkyl inwhich each of the rings shares two adjacent atoms with the other ring.The second ring of a fused bicyclic cycloalkyl may be selected fromsaturated, unsaturated and aromatic rings. A “cycloalkenyl” group is acyclic hydrocarbon containing one or more double bonds.

The term “(cycloalkyl)alkyl”, as used herein, refers to an alkyl groupsubstituted with a cycloalkyl group.

The term “carbonate” is art-recognized and refers to a group —CO₂—R¹⁰,wherein R¹⁰ represents a hydrocarbyl group.

The term “carboxy”, as used herein, refers to a group represented by theformula —CO₂H.

The term “ester”, as used herein, refers to a group —C(O)OR¹⁰ whereinR¹⁰ represents a hydrocarbyl group.

The term “ether”, as used herein, refers to a hydrocarbyl group linkedthrough an oxygen to another hydrocarbyl group. Accordingly, an ethersubstituent of a hydrocarbyl group may be hydrocarbyl-O—. Ethers may beeither symmetrical or unsymmetrical. Examples of ethers include, but arenot limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethersinclude “alkoxyalkyl” groups, which may be represented by the generalformula alkyl-O-alkyl.

The terms “halo” and “halogen” as used herein means halogen and includeschloro, fluoro, bromo, and iodo.

The term “heteroaralkyl”, as used herein, refers to an alkyl groupsubstituted with a heteroaryl group.

The term “heteroalkyl”, as used herein, refers to a saturated orunsaturated chain of carbon atoms and at least one heteroatom, whereinno two heteroatoms are adjacent.

The term “heteroaryl” includes substituted or unsubstituted aromaticsingle ring structures, preferably 5- to 7-membered rings, morepreferably 5- to 6-membered rings, whose ring structures include atleast one heteroatom, preferably one to four heteroatoms, morepreferably one or two heteroatoms. The terms “heteroaryl” also includepolycyclic ring systems having two or more cyclic rings in which two ormore carbons are common to two adjoining rings wherein at least one ofthe rings is heteroaromatic, e.g., the other cyclic rings can becycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls. Heteroaryl groups include, for example, pyrrole, furan,thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine,pyridazine, and pyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, andsulfur.

The terms “heterocycloalkyl”, “heterocycle”, and “heterocyclic” refer tosubstituted or unsubstituted non-aromatic ring structures, preferably 3-to 10-membered rings, more preferably 3- to 7-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heterocycloalkyl” and “heterocyclic” also include polycyclic ringsystems having two or more cyclic rings in which two or more carbons arecommon to two adjoining rings wherein at least one of the rings isheterocyclic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Heterocycloalkyl groups include, for example, piperidine, piperazine,pyrrolidine, morpholine, lactones, lactams, and the like.

The term “(heterocycloalkyl)alkyl”, as used herein, refers to an alkylgroup substituted with a heterocycloalkyl group.

The term “hydrocarbyl”, as used herein, refers to a group that is bondedthrough a carbon atom that does not have a ═O or ═S substituent, andtypically has at least one carbon-hydrogen bond and a primarily carbonbackbone, but may optionally include heteroatoms. Thus, groups likemethyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to behydrocarbyl for the purposes of this application, but substituents suchas acetyl (which has a ═O substituent on the linking carbon) and ethoxy(which is linked through oxygen, not carbon) are not. Hydrocarbyl groupsinclude, but are not limited to aryl, heteroaryl, carbocycle,heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.

The term “hydroxyalkyl”, as used herein, refers to an alkyl groupsubstituted with a hydroxy group.

The term “lower” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups where there are ten or fewer non-hydrogen atoms in thesubstituent, preferably six or fewer. A “lower alkyl”, for example,refers to an alkyl group that contains ten or fewer carbon atoms,preferably six or fewer. In certain embodiments, acyl, acyloxy, alkyl,alkenyl, alkynyl, or alkoxy substituents defined herein are respectivelylower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, orlower alkoxy, whether they appear alone or in combination with othersubstituents, such as in the recitations hydroxyalkyl and aralkyl (inwhich case, for example, the atoms within the aryl group are not countedwhen counting the carbon atoms in the alkyl substituent).

The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two ormore rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls) in which two or more atoms are commonto two adjoining rings, e.g., the rings are “fused rings”. Each of therings of the polycycle can be substituted or unsubstituted. In certainembodiments, each ring of the polycycle contains from 3 to 10 atoms inthe ring, preferably from 5 to 7.

The term “silyl” refers to a silicon moiety with three hydrocarbylmoieties attached thereto.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include any substituents described herein,for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine,an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that substituents canthemselves be substituted, if appropriate. Unless specifically stated as“unsubstituted,” references to chemical moieties herein are understoodto include substituted variants. For example, reference to an “aryl”group or moiety implicitly includes both substituted and unsubstitutedvariants.

The term “sulfate” is art-recognized and refers to the group —OSO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfonamide” is art-recognized and refers to the grouprepresented by the general formulae

wherein R⁹ and R¹⁰ independently represents hydrogen or hydrocarbyl,such as alkyl, or R⁹ and R¹⁰ taken together with the intervening atom(s)complete a heterocycle having from 4 to 8 atoms in the ring structure.

The term “sulfoxide” is art-recognized and refers to the group—S(O)—R¹⁰, wherein R¹⁰ represents a hydrocarbyl.

The term “sulfonate” is art-recognized and refers to the group SO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfone” is art-recognized and refers to the group —S(O)₂—R¹⁰,wherein R¹⁰ represents a hydrocarbyl.

The term “thioalkyl”, as used herein, refers to an alkyl groupsubstituted with a thiol group.

The term “thioester”, as used herein, refers to a group —C(O)SR¹⁰ or—SC(O)R¹⁰ wherein R¹⁰ represents a hydrocarbyl.

The term “thioether”, as used herein, is equivalent to an ether, whereinthe oxygen is replaced with a sulfur.

The term “urea” is art-recognized and may be represented by the generalformula

wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbyl,such as alkyl, or either occurrence of R⁹ taken together with R¹⁰ andthe intervening atom(s) complete a heterocycle having from 4 to 8 atomsin the ring structure.

The term “dioxaborolane”, as used herein, refers to a chemical grouprepresented by the general formula:

wherein the dioaxaborolane is optionally substituted at anysubstitutable position by one or more substituents including, but notlimited to, alkyl (e.g., substituted alkyl), hydroxyalkyl, alkoxyl,carboxyalkyl, —COOH, aryl, heteroaryl, aralkyl, heteroaralkyl, etc.Alternatively, the dioxaborolane can be substituted at two adjacentsubstitutable positions, such that the two substituents, together withthe intervening atoms, form an optionally substituted cycloalkyl or arylring (as in, e.g., catecholatoboron-).

The term “dioxaborolanone”, as used herein, refers to a chemical grouprepresented by the general formula:

wherein the dioxaborolanone is optionally substituted at anysubstitutable position by one or more substituents including, but notlimited to, alkyl (e.g., substituted alkyl), hydroxyalkyl, alkoxyl,carboxyalkyl, —COOH, aryl, heteroaryl, aralkyl, heteroaralkyl, etc.

The term “dioxaborolandione”, as used herein, refers to a chemical grouprepresented by the general formula:

The term “dioxaborinane”, as used herein, refers to a chemical grouprepresented by the general formula:

wherein the dioxaborinane is optionally substituted at any substitutableposition by one or more substituents including, but not limited to,alkyl (e.g., substituted alkyl), hydroxyalkyl, alkoxyl, carboxyalkyl,—COOH, aryl, heteroaryl, aralkyl, heteroaralkyl, etc. Alternatively, thedioxaborinane can be substituted at two adjacent substitutablepositions, such that the two substituents, together with the interveningatoms, form an optionally substituted cycloalkyl or aryl ring.

The term “dioxaborinanone”, as used herein, refers to a chemical grouprepresented by the general formula:

wherein the dioxaborinanone is optionally substituted at anysubstitutable position by one or more substituents including, but arenot limited to, alkyl (e.g., substituted alkyl), hydroxyalkyl, alkoxyl,carboxyalkyl, —COOH, aryl, heteroaryl, aralkyl, heteroaralkyl, etc.

The term “dioxaborinandione”, as used herein, refers to a chemical grouprepresented by the general formula:

wherein the dioxaborinandione is optionally substituted at anysubstitutable position by one or more substituents including, but arenot limited to, alkyl (e.g., substituted alkyl), hydroxyalkyl, alkoxyl,carboxyalkyl, —COOH, aryl, heteroaryl, aralkyl, heteroaralkyl, etc.

“Protecting group” refers to a group of atoms that, when attached to areactive functional group in a molecule, mask, reduce or prevent thereactivity of the functional group. Typically, a protecting group may beselectively removed as desired during the course of a synthesis.Examples of protecting groups can be found in Greene and Wuts,Protective Groups in Organic Chemistry, 3^(rd) Ed., 1999, John Wiley &Sons, NY and Harrison et al., Compendium of Synthetic Organic Methods,Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Representative nitrogenprotecting groups include, but are not limited to, formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl(“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl(“TES”), trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl(“NVOC”) and the like. Representative hydroxyl protecting groupsinclude, but are not limited to, those where the hydroxyl group iseither acylated (esterified) or alkylated such as benzyl and tritylethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilylethers (e.g., TMS or TIPS groups), glycol ethers, such as ethyleneglycol and propylene glycol derivatives and allyl ethers.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample.

The term “treating” includes prophylactic and/or therapeutic treatments.The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The term “prodrug” is intended to encompass compounds which, underphysiologic conditions, are converted into the therapeutically activeagents of the present invention (e.g., a compound of Formula I, Ia, Ib,Ic, Id, Ie, If, Ig, or Ih). A common method for making a prodrug is toinclude one or more selected moieties which are hydrolyzed underphysiologic conditions to reveal the desired molecule. In otherembodiments, the prodrug is converted by an enzymatic activity of thehost animal. For example, esters or carbonates (e.g., esters orcarbonates of alcohols or carboxylic acids) are preferred prodrugs ofthe present invention. Alternatively, amides (e.g., an amide of an aminogroup) may be a prodrug of the invention. In certain embodiments, someor all of the compounds of formula I in a formulation represented abovecan be replaced with the corresponding suitable prodrug, e.g., wherein ahydroxyl in the parent compound is presented as an ester or a carbonateor carboxylic acid present in the parent compound is presented as anester.

In other preferred embodiments, prodrugs of the invention encompasscompounds in which the boronic acid is esterified or otherwise modifiedto form a boronic acid derivative capable of hydrolyzing underphysiologic conditions to the parent boronic acid. For example, thecompounds of the invention include tartrate or citrate “esters” ofboronic acids, including where the boron forms a boracycle by bonding totwo heteroatoms of the tartrate or citrate moiety. Analogously, thecompounds of the invention include mandelic acid or oxalic acid estersof the parent boronic acids. Representative boronic acid esters arepictured below:

Pharmaceutical Compositions

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising a compound of the invention, such as a compoundof formula I, Ia, Ib, Ic, Id, Ie, If, Ig, or Ih, or a pharmaceuticallyacceptable salt thereof; and a pharmaceutically acceptable carrier.

In certain embodiments, the present invention provides a pharmaceuticalpreparation suitable for use in a human patient, comprising any compoundof the invention (e.g., a compound of formula I, Ia, Ib, Ic, Id, Ie, If,Ig, or Ih), and one or more pharmaceutically acceptable excipients. Incertain embodiments, the pharmaceutical preparations may be for use intreating or preventing a condition or disease as described herein. Incertain embodiments, the pharmaceutical preparations have a low enoughpyrogen activity to be suitable for use in a human patient.

One embodiment of the present invention provides a pharmaceutical kitcomprising a compound of the invention, such as a compound of formula I,Ia, Ib, Ic, Id, Ie, If, Ig, or Ih, or a pharmaceutically acceptable saltthereof, and optionally directions on how to administer the compound.

The compositions and methods of the present invention may be utilized totreat an individual in need thereof. In certain embodiments, theindividual is a mammal such as a human, or a non-human mammal. Whenadministered to an animal, such as a human, the composition or thecompound is preferably administered as a pharmaceutical compositioncomprising, for example, a compound of the invention and apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are well known in the art and include, for example, aqueoussolutions such as water or physiologically buffered saline or othersolvents or vehicles such as glycols, glycerol, oils such as olive oil,or injectable organic esters. In certain preferred embodiments, whensuch pharmaceutical compositions are for human administration,particularly for invasive routes of administration (i.e., routes, suchas injection or implantation, that circumvent transport or diffusionthrough an epithelial barrier), the aqueous solution is pyrogen-free, orsubstantially pyrogen-free. The excipients can be chosen, for example,to effect delayed release of an agent or to selectively target one ormore cells, tissues or organs. The pharmaceutical composition can be indosage unit form such as tablet, capsule (including sprinkle capsule andgelatin capsule), granule, lyophile for reconstitution, powder,solution, syrup, suppository, injection or the like. The composition canalso be present in a transdermal delivery system, e.g., a skin patch.The composition can also be present in a solution suitable for topicaladministration, such as an eye drop.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable agents that act, for example, to stabilize, increasesolubility or to increase the absorption of a compound such as acompound of the invention. Such physiologically acceptable agentsinclude, for example, carbohydrates, such as glucose, sucrose ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins or other stabilizers orexcipients. The choice of a pharmaceutically acceptable carrier,including a physiologically acceptable agent, depends, for example, onthe route of administration of the composition. The preparation orpharmaceutical composition can be a selfemulsifying drug delivery systemor a selfmicroemulsifying drug delivery system. The pharmaceuticalcomposition (preparation) also can be a liposome or other polymermatrix, which can have incorporated therein, for example, a compound ofthe invention. Liposomes, for example, which comprise phospholipids orother lipids, are nontoxic, physiologically acceptable and metabolizablecarriers that are relatively simple to make and administer.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

A pharmaceutical composition (preparation) can be administered to asubject by any of a number of routes of administration including, forexample, orally (for example, drenches as in aqueous or non-aqueoussolutions or suspensions, tablets, capsules (including sprinkle capsulesand gelatin capsules), boluses, powders, granules, pastes forapplication to the tongue); absorption through the oral mucosa (e.g.,sublingually); anally, rectally or vaginally (for example, as a pessary,cream or foam); parenterally (including intramuscularly, intravenously,subcutaneously or intrathecally as, for example, a sterile solution orsuspension); nasally; intraperitoneally; subcutaneously; transdermally(for example as a patch applied to the skin); and topically (forexample, as a cream, ointment or spray applied to the skin, or as an eyedrop). The compound may also be formulated for inhalation. In certainembodiments, a compound may be simply dissolved or suspended in sterilewater. Details of appropriate routes of administration and compositionssuitable for same can be found in, for example, U.S. Pat. Nos.6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and4,172,896, as well as in patents cited therein.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient that can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association an active compound, such as a compound ofthe invention, with the carrier and, optionally, one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association a compound of the present inventionwith liquid carriers, or finely divided solid carriers, or both, andthen, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules (including sprinkle capsules and gelatin capsules),cachets, pills, tablets, lozenges (using a flavored basis, usuallysucrose and acacia or tragacanth), lyophile, powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of a compound of the present invention as anactive ingredient. Compositions or compounds may also be administered asa bolus, electuary or paste.

To prepare solid dosage forms for oral administration (capsules(including sprinkle capsules and gelatin capsules), tablets, pills,dragees, powders, granules and the like), the active ingredient is mixedwith one or more pharmaceutically acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; (10) complexing agents,such as, modified and unmodified cyclodextrins; and (11) coloringagents. In the case of capsules (including sprinkle capsules and gelatincapsules), tablets and pills, the pharmaceutical compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-filled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions, such as dragees, capsules (including sprinkle capsules andgelatin capsules), pills and granules, may optionally be scored orprepared with coatings and shells, such as enteric coatings and othercoatings well known in the pharmaceutical-formulating art. They may alsobe formulated so as to provide slow or controlled release of the activeingredient therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile, otherpolymer matrices, liposomes and/or microspheres. They may be sterilizedby, for example, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions that can be dissolved in sterile water, or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions that can be used includepolymeric substances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Liquid dosage forms useful for oral administration includepharmaceutically acceptable emulsions, lyophiles for reconstitution,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, cyclodextrins and derivatives thereof, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ (e.g., wheat germ), olive, castor and sesame oils), glycerol,tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters ofsorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions for rectal, vaginal, orurethral administration may be presented as a suppository, which may beprepared by mixing one or more active compounds with one or moresuitable nonirritating excipients or carriers comprising, for example,cocoa butter, polyethylene glycol, a suppository wax or a salicylate,and which is solid at room temperature, but liquid at body temperatureand, therefore, will melt in the rectum or vaginal cavity and releasethe active compound.

Formulations of the pharmaceutical compositions for administration tothe mouth may be presented as a mouthwash, or an oral spray, or an oralointment.

Alternatively or additionally, compositions can be formulated fordelivery via a catheter, stent, wire, or other intraluminal device.Delivery via such devices may be especially useful for delivery to thebladder, urethra, ureter, rectum, or intestine.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the active compound in theproper medium. Absorption enhancers can also be used to increase theflux of the compound across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.Exemplary ophthalmic formulations are described in U.S. Publication Nos.2005/0080056, 2005/0059744, 2005/0031697 and 2005/004074 and U.S. Pat.No. 6,583,124, the contents of which are incorporated herein byreference. If desired, liquid ophthalmic formulations have propertiessimilar to that of lacrimal fluids, aqueous humor or vitreous humor orare compatable with such fluids. A preferred route of administration islocal administration (e.g., topical administration, such as eye drops,or administration via an implant).

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

Pharmaceutical compositions suitable for parenteral administrationcomprise one or more active compounds in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

For use in the methods of this invention, active compounds can be givenper se or as a pharmaceutical composition containing, for example, 0.1to 99.5% (more preferably, 0.5 to 90%) of active ingredient incombination with a pharmaceutically acceptable carrier.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinacious biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a compound at a particular targetsite.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient that is effective to achieve the desired therapeutic responsefor a particular patient, composition, and mode of administration,without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound or combination ofcompounds employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound(s) being employed, the duration of the treatment,other drugs, compounds and/or materials used in combination with theparticular compound(s) employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the therapeutically effective amount of thepharmaceutical composition required. For example, the physician orveterinarian could start doses of the pharmaceutical composition orcompound at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. By “therapeutically effective amount” ismeant the concentration of a compound that is sufficient to elicit thedesired therapeutic effect. It is generally understood that theeffective amount of the compound will vary according to the weight, sex,age, and medical history of the subject. Other factors which influencethe effective amount may include, but are not limited to, the severityof the patient's condition, the disorder being treated, the stability ofthe compound, and, if desired, another type of therapeutic agent beingadministered with the compound of the invention. A larger total dose canbe delivered by multiple administrations of the agent. Methods todetermine efficacy and dosage are known to those skilled in the art(Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13ed., 1814-1882, herein incorporated by reference).

In general, a suitable daily dose of an active compound used in thecompositions and methods of the invention will be that amount of thecompound that is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above.

If desired, the effective daily dose of the active compound may beadministered as one, two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In certain embodiments of the presentinvention, the active compound may be administered two or three timesdaily. In preferred embodiments, the active compound will beadministered once daily.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

In certain embodiments, compounds of the invention may be used alone orconjointly administered with another type of therapeutic agent. As usedherein, the phrase “conjoint administration” refers to any form ofadministration of two or more different therapeutic compounds such thatthe second compound is administered while the previously administeredtherapeutic compound is still effective in the body (e.g., the twocompounds are simultaneously effective in the patient, which may includesynergistic effects of the two compounds). For example, the differenttherapeutic compounds can be administered either in the same formulationor in a separate formulation, either concomitantly or sequentially. Incertain embodiments, the different therapeutic compounds can beadministered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72hours, or a week of one another. Thus, an individual who receives suchtreatment can benefit from a combined effect of different therapeuticcompounds.

In certain embodiments, conjoint administration of compounds of theinvention with one or more additional therapeutic agent(s) (e.g., one ormore additional chemotherapeutic agent(s)) provides improved efficacyrelative to each individual administration of the compound of theinvention (e.g., compound of formula I, Ia, Ib, Ic, Id, Ie, If, Ig, orIh) or the one or more additional therapeutic agent(s). In certain suchembodiments, the conjoint administration provides an additive effect,wherein an additive effect refers to the sum of each of the effects ofindividual administration of the compound of the invention and the oneor more additional therapeutic agent(s).

This invention includes the use of pharmaceutically acceptable salts ofcompounds of the invention in the compositions and methods of thepresent invention. The term “pharmaceutically acceptable salt” as usedherein includes salts derived from inorganic or organic acids including,for example, hydrochloric, hydrobromic, sulfuric, nitric, perchloric,phosphoric, formic, acetic, lactic, maleic, fumaric, succinic, tartaric,glycolic, salicylic, citric, methanesulfonic, benzenesulfonic, benzoic,malonic, trifluoroacetic, trichloroacetic, naphthalene-2-sulfonic,oxalic, mandelic and other acids. Pharmaceutically acceptable salt formscan include forms wherein the ratio of molecules comprising the salt isnot 1:1. For example, the salt may comprise more than one inorganic ororganic acid molecule per molecule of base, such as two hydrochloricacid molecules per molecule of compound of Formula I, Ia, Ib, Ic, Id, e,If, Ig, or Ih. As another example, the salt may comprise less than oneinorganic or organic acid molecule per molecule of base, such as twomolecules of compound of Formula I, Ia, Ib, Ic, Id, e, If, Ig, or Ih permolecule of tartaric acid.

In further embodiments, contemplated salts of the invention include, butare not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammoniumsalts. In certain embodiments, contemplated salts of the inventioninclude, but are not limited to, L-arginine, benenthamine, benzathine,betaine, calcium hydroxide, choline, deanol, diethanolamine,diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine,N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine,magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium,1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine,and zinc salts. In certain embodiments, contemplated salts of theinvention include, but are not limited to, Na, Ca, K, Mg, Zn or othermetal salts.

The pharmaceutically acceptable acid addition salts can also exist asvarious solvates, such as with water, methanol, ethanol,dimethylformamide, and the like. Mixtures of such solvates can also beprepared. The source of such solvate can be from the solvent ofcrystallization, inherent in the solvent of preparation orcrystallization, or adventitious to such solvent.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1)water-soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3)metal-chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

EXAMPLES Example 1: Synthetic Methods

The scheme below and subsequect experimental procedures illustrates ageneral method that can be used to prepare examples included in theinvention. Variations in the method may be preferable depending on thesalt form desired. For example, if the hydrochloric acid salt isdesired, intermediate 8 can be treated with hydrogen gas in the presenceof palladium on carbon to give intermediate amino acid 9. Subsequenttreatment with aqueous hydrochloric acid gives the target arginaseinhibitor 10 as the hydrochloric acid salt.

If the free-base is desired, intermediate 8 can be used in a modifiedprocedure. Here, treatment with trifluoroacetic acid followed byisobutylboronic acid gives intermediate amine 12. Subsequent reductionof the azide and deprotection of the benzyl ester using hydrogen gas inthe presence of palladium on carbon gives target arginase inhibitor 13as a free-base. A detailed description of these methods is providedbelow.

Synthesis of(3R,4S)-3-amino-1-((S)-2-aminopropanoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylicacid, dihydrochloride salt (10)

Step 1: Synthesis oftert-Butyl-trans-3-allyl-4-hydroxypyrrolidine-1-carboxylate (2, racemic)

Allyl magnesium bromide (1,037 mL, 713 mmol, 0.69 M in diethyl ether)was cooled to 0° C. and carefully treated with tert-butyl6-oxa-3-azabicyclo[3.1.0]hexane-3-carboxylate (60 g, 323.9 mmol) inanhydrous diethyl ether (324 mL, 1 M). After the addition was complete,the reaction mixture was stirred for 15 min, slowly quenched withsaturated aqueous ammonium chloride (500 mL), extracted with diethylether (2×400 mL), dried over MgSO₄, filtered and concentrated.Purification by flash column chromatography (20-40% ethyl acetate inheptane) gavetert-butyl-trans-3-allyl-4-hydroxypyrrolidine-1-carboxylate (2, 64.33 g,87% yield) as a pale yellow oil. ¹H-NMR (CDCl₃, 400 MHz): δ_(H): 5.80(1H, m), 5.06 (2H, m), 4.07 (1H, m), 3.57 (2H, m), 3.22 (1H, m), 3.08(1H, m), 2.26-2.10 (2H, m) and 1.45 (9H, s).

Step 2: Synthesis of tert-Butyl-3-allyl-4-oxopyrrolidine-1-carboxylate(3, racemic)

While under an atmosphere of dry nitrogen, an ice-cooled solution oftert-butyl-trans-3-allyl-4-hydroxypyrrolidine-1-carboxylate (2, 60 g,264 mmol) and diisopropylethylamine (132.2 mL, 799.8 mmol) indichloromethane (750 mL, 0.35 M) was treated dropwise with a solution ofsulfur trioxide pyridine complex (94.95 g, 596.6 mmol) in anhydrous DMSO(750 mL) at a rate to keep the reaction mixture below 10° C. After theaddition was complete, the mixture was stirred at 3° C. for 15 min,quenched with water (380 mL) and extracted with ethyl acetate (500 mL,then 2×300 mL). The combined organic solution was washed twice withwater (200 mL), once with saturated aqueous sodium chloride (200 mL),dried (MgSO₄) and concentrated. The resulting crude oil was distilled at105° C. (0.4 mm Hg) to afford tert-butyl3-allyl-4-oxopyrrolidine-1-carboxylate (3, 58 g, 83% yield) as acolorless oil. H-NMR (CDCl₃, 400 MHz): δ_(H): 5.74 (1H, m), 5.09 (2H,m), 4.02 (1H, m), 3.88 (1H, d, J=19.4 Hz), 3.68 (1H, d, J=19.4 Hz), 3.31(1H, dd, J=9.4, 8.3 Hz), 2.65 (1H, m), 2.54 (1H, m), 2.18 (1H, m) and1.45 (9H, s).

Step 3: Synthesis oftrans-4-Allyl-3-azido-1-(tert-butoxycarbonyl)pyrrolidine-3-carboxylicacid (4, racemic)

A solution of chloroform (26.86 mL, 333 mmol) and TMS-Cl (32.86 mL,257.1 mmol) in anhydrous THF (300 mL) was cooled to −78° C. Afterstirring for 10 min, LHMDS (1M in THF, 249 mL, 249 mmol) was added at arate such that the temperature remained below −60° C. (approximately 30min). After stirring an additional 30 min at −60 to −70° C. (reactionmixture becomes cloudy) the solution was warmed to −20° C. (reactionmixture becomes clear) and treated withtert-butyl-3-allyl-4-oxopyrrolidine-1-carboxylate (3, 30 g, 133.2 mmol)in DMF (90 mL) and tetrabutylammonium acetate (3.69 g, 12.24 mmol) inDMF (90 mL) at a rate such that the internal reaction temperatureremained below −20° C. (reaction becomes cloudy). After the addition wascomplete, the reaction mixture was warmed to room temperature withstirring until the ketone starting material was consumed (by TLC), thenpoured into saturated aqueous NH₄Cl and extracted with EtOAc (3×100 mL).The combined organic layers were washed successively with saturatedaqueous NH₄Cl and saturated aqueous NaCl (2×80 mL), dried over MgSO₄,filtered and concentrated.

While under nitrogen, the crude TMS protected intermediate was dissolvedin dry THF (300 mL), cooled to 0° C. and carefully treated with aceticacid (7.5 mL, 130.9 mmol) and TBAF (1 M in THF, 133.2 mL, 133.2 mmol)dropwise. After the addition was complete, the reaction was stirred anadditional 10 min at 0° C. then poured into saturated aqueous NaHCO₃ andextracted with EtOAc (3×100 mL). The combined organic layers were washedwith saturated aqueous NaCl, dried over MgSO₄, filtered and concentratedto afford the crude alcohol intermediate.

The crude alcohol was dissolved in dioxane (200 mL), cooled to 0° C.,and treated with a pre-cooled (0° C.) solution of sodium azide (14.04 g,399.5 mmol) and NaOH (15.98 g, 399.5 mmol) in water (200 mL) dropwise.The resulting reaction mixture was allowed to warm to room temperaturewith stirring overnight then quenched with of saturated aqueous NH₄Cland was extracted with EtOAc (500 mL). The aqueous layer was separatedand extracted with EtOAc (2×300 mL). The combined organic layers werewashed with water and saturated aqueous NaCl, dried over MgSO₄, filteredand concentrated to give crudetrans-4-allyl-3-azido-1-(tert-butoxycarbonyl)pyrrolidine-3-carboxylicacid (4, crude 45 g) which was used without further purification. ¹H-NMR(CDCl₃, 400 MHz): δ_(H): 5.80 (1H, m), 5.06 (2H, m), 4.05 (1H, dd,J=9.9, 4.9 Hz), 3.59 (2H, m), 3.22 (1H, dd, J=11.6, 4.4 Hz), 3.08 (1H,dd, J=11.0, 5.2 Hz), 2.24-2.04 (2H, m), 1.65 (1H, br s, OH) and 1.45(9H, s).

Step 4: Synthesis oftrans-3-Benzyl-1-(tert-butyl)-4-allyl-3-azidopyrrolidine-1,3-dicarboxylate

A solution of crudetrans-4-allyl-3-azido-1-(tert-butoxycarbonyl)pyrrolidine-3-carboxylicacid (4, 39.5 g, 133 mmol—calculated quantity assuming 100% yield fromprevious steps) and K₂CO₃ (92.04 g, 666 mmol) in acetonitrile (317 mL)was cooled to 0° C. and treated with benzyl bromide (17.52 mL, 146.5mmol). After stirring overnight at room temperature the solution wasconcentrated, dissolved in EtOAc (600 mL), washed with saturated aqueousNaCl, dried over MgSO₄, filtered and concentrated. Purification viasilica gel chromatography (10 to 30% EtOAc in hexane) gavetrans-3-benzyl-1-(tert-butyl)-4-allyl-3-azidopyrrolidine-1,3-dicarboxylateas yellow liquid (5, 40 g, 78% yield).

The product was separated into its enantiomers using a ChiralTechnologies Chiralpak ADH column with isopropyl alcohol and hexanes(2:98) as an eluent. Analysis of the separated enantiomers using ananalytical Chiralpak ADH column (4.6×250 mm) with the same eluent and aflow rate of 1.0 mL/min and UV detection (210 nm) gave the desiredenantiomer (3-benzyl-1-(tert-butyl)(3R,4S)-4-allyl-3-azidopyrrolidine-1,3-dicarboxylate, 5a) with aretention time of 13.5 min and the undesired enantiomer(3-benzyl-1-(tert-butyl)(3S,4R)-4-allyl-3-azidopyrrolidine-1,3-dicarboxylate, 5b) at 10.3 min,each with an enantiomeric excess of approximately 98%. ¹H-NMR (CDCl₃,400 MHz): δ_(H): 7.37 (5H, s), 5.62 (1H, m), 5.25 (2H, m), 5.00 (2H, m),3.88 (1H, dd, J=37.2, 12.0 Hz), 3.58 (1H, ddd, J=37.2, 11.0, 7.0 Hz),3.42 (1H, dd, J=21.4, 12.0 Hz), 3.28 (1H, ddd, J=28.3, 11.0, 5.4 Hz),2.41 (1H, m), 2.11 (1H, m), 1.80 (1H, m) and 1.44 (9H, s).

Step 5: Synthesis of (3R,4S)-3-Benzyl 1-tert-butyl3-azido-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-1,3-dicarboxylate(6)

A stirred solution of 3-benzyl-1-(tert-butyl)(3R,4S)-4-allyl-3-azidopyrrolidine-1,3-dicarboxylate (5a, 16.4 g, 42.4mmol) in anhydrous methylene chloride (130 mL), under an atmosphere ofnitrogen, was treated with bis(1,5-cyclooctadiene)diiridium(I)dichloride (0.75 g, 1.12 mmol) and 1,2-bis(diphenylphosphino)ethane(0.894 g, 2.24 mmol) and the reaction was stirred for 30 minutes at roomtemperature and then cooled to −25° C.4,4,5,5-tetramethyl[1,3,2]dioxaborolane (9.83 mL, 67.75 mmol) was addeddropwise and then the reaction was allowed to slowly warm to roomtemperature and stirred for 20 hrs. Water (60 mL) was added and thereaction was stirred for 10 minutes, and then the methylene chloride wasremoved under reduced pressure. The remaining aqueous phase wasextracted with ethyl acetate (3×100 mL). The combined organic phase waswashed with brine, dried over anhydrous magnesium sulfate, filtered andconcentrated in vacuo. The residual solid was passed through a short padof silica gel, eluting with 15% to 30% ethyl acetate in hexane, to give(3R,4S)-3-benzyl 1-tert-butyl3-azido-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-1,3-dicarboxylate(6, 12.5 g, 57%). ¹H-NMR (CDCl₃, 400 MHz): δ_(H): 7.35 (5H, m), 5.23(2H, m), 3.85 (1H, dd, J=39.3, 11.8 Hz), 3.60 (1H, m), 3.37 (1H, dd,J=24.3, 11.8 Hz), 3.25 (1H, ddd, J=40, 10.6, 6.6 Hz), 2.33 (1H, m), 1.43(9H, s), 1.39-1.26 (3H, m), 1.21 (12H, s), 1.07 (1H, m) and 0.68 (2H,m).

Step 6: Synthesis of(3R,4S)-3-Benzyl-3-azido-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-3-carboxylate,trifluoroacetic acid salt (7)

A solution of (3R,4S)-3-benzyl 1-tert-butyl3-azido-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-1,3-dicarboxylate(6, 10.2 g, 19.8 mmol) was dissolved in anhydrous methylene chloride(160 mL), cooled to 0° C. and treated with trifluoroacetic acid (40 mL).The reaction mixture was then allowed to warm, stirred at roomtemperature for 4 hr and then concentrated under reduced pressure togive a viscous oil. The resultant oil was azeotroped with dry toluene(3×100 mL) to remove residual trifluoroacetic acid and then dried underhigh vacuum to give(3R,4S)-3-benzyl-3-azido-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-3-carboxylate,trifluoroacetic acid salt (7) as a very viscous oil (10.56 g), whichslowly turns to a glass. ¹H-NMR (CDCl₃, 400 MHz): δ_(H): 9.7 (1H, br m(exch), NH), 7.55 (1H, br s (exch), NH), 7.38 (5H, m), 5.31 (1H, d,J=11.7 Hz), 5.26 (1H, d, J=11.7 Hz), 3.77 (1H, d, J=12.5 Hz), 3.65 (1H,dd, J=11.8, 7.8 Hz), 3.32 (1H, d, J=12.4 Hz), 3.18 (1H, m), 2.54 (1H,m), 1.45-1.26 (3H, m), 1.22 (12H, s), 1.02 (1H, m) and 0.63 (2H, t,J=7.4 Hz).

Step 7: Synthesis of(3R,4S)-3-Benzyl-3-azido-1-((S)-2-((tert-butoxylcarbonyl)amino)propanyoyl)-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-3-carboxylate(8)

To a stirred solution of(3R,4S)-3-benzyl-3-azido-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-3-carboxylate,trifluoroacetic acid salt (7, 10.56 g, 19.8 mmol) in anhydrous methylenechloride (150 mL) was added DMAP (50 mg, catalytic) and HOBt (50 mg,catalytic) and N-(tert-butoxycarbonyl)-L-alanine (5.62 g, 29.7 mmol).The reaction was cooled to 0° C. under an atmosphere of dry nitrogen andthen treated with EDCI (5.69 g, 29.7 mmol) and triethylamine (8.3 mL,59.4 mmol). The reaction was stirred at 0° C. for 1 hr and then allowedto warm to room temperature and stirred for 16 hrs at this temperature.The reaction was poured into water (100 mL), stirred for 20 mins andthen the phases were separated. The aqueous phase was extracted with3×50 mL methylene chloride. The combined organic phase was washed withwater, 1 N hydrochloric acid and brine, then dried over anhydrousmagnesium sulfate, filtered and concentrated in vacuo. The residual oilwas passed through a pad of silica gel, eluting with 5% to 50% ethylacetate in hexanes, to give(3R,4S)-3-benzyl-3-azido-1-((S)-2-((tert-butoxylcarbonyl)amino)propanyoyl)-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-3-carboxylate(8) as a colorless oil (9.50 g, 82%), seen as a 1:1 mixture of rotamersby NMR at room temperature; ¹H-NMR (CDCl₃, 400 MHz): δ_(H):7.56 (5H, m),5.40 (0.5H, d, J=8.0 Hz, NH) and 5.34 (0.5H, d, J=8.0 Hz, NH), 5.29-5.19(2H, m), 4.39 (0.5H, dq, J=7.2, 7.0 Hz) and 4.30 (0.5H, dq, J=7.2, 7.0Hz), 4.06 (0.5H, d, J=13.0 Hz) and 3.89 (0.5H, d, J=11.1 Hz), 3.81(0.5H, dd, J=12.0, 7.3 Hz) and 3.69 (0.5H, J=10.0, 7.0 Hz), 3.61 (0.5H,d, J=11.1 Hz) and 3.47 (0.5H, d, J=13.0 Hz), 3.54 (0.5H, dd, J=10.0, 6.0Hz) and 3.33 (0.5H, dd, J=12.0, 6.3 Hz), 2.41 (1H, m), 1.43 (4.5H, s)and 1.42 (4.5H, s), 1.40-1.28 (3H, m), 1.31 (1.5H, d, J=6.8 Hz) and 1.20(1.5H, J=6.8 Hz), 1.22 (12H, s), 1.04 (1H, m) and 0.67 (2H, m).

Step 8: Synthesis of(3R,4S)-3-amino-1-((S)-2-((tert-butoxylcarbonyl)amino)propanyoyl)-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-3-carboxylic acid (9)

(3R,4S)-3-benzyl-3-azido-1-((S)-2-((tert-butoxylcarbonyl)amino)propanyoyl)-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-3-carboxylate(8, 9.48 g, 16.2 mmol) was dissolved in a 1:1 mixture of ethyl acetateand ethanol (120 mL). 10% Palladium on charcoal (500 mg) was added andthe solution was degassed under vacuum and purged with hydrogen(hydrogen balloon). This purging procedure was repeated 3× and then thereaction was stirred under a hydrogen atmosphere for 5 hours. Thereaction was placed back under vacuum to remove the residual hydrogenand then filtered through a pad of celite, with 4×30 mL ethanol washes.The solution was concentrated to 20 mL under vacuum and then filteredthrough a 4μ syringe filter to remove traces of palladium. The solutionwas concentrated to dryness under vacuum and used without furtherpurification. LC-MS: ESI+ (0.1% HCOOH in IPA/water): m/z forC₂₂H₄₀BN₃O₇: expected 469.3, observed 492.3 (M+Na)⁺, 470.3 (M+H)⁺, 414.2(M+H−^(i)Bu)⁺, 370.3 (M+H−Boc)⁺, ESI−: 468.0 (M−H)⁻.

Step 9: Synthesis of(3R,4S)-3-Amino-1-((S)-2-aminopropanyoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylicacid, dihydrochloride salt (10)

A suspension of(3R,4S)-3-amino-1-((S)-2-((tert-butoxylcarbonyl)amino)propanyoyl)-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-3-carboxylicacid (from the previous step) in 4N hydrochloric acid (50 mL) wasstirred at 50° C. for 16 hrs and then cooled to room temperature. Thereaction was diluted with a further 50 mL of water and then washed 5×with methylene chloride. The aqueous phase was concentrated to drynessunder reduced pressure, keeping the water bath at or below 50° C. Theresultant oil was dissolved in water (30 mL) and concentrated. Thisprocedure was repeated 2× with further 30 mL aliquots of water and thendried under vacuum to give a pale yellow foam.

Dowex 550A-UPW hydroxide resin (75 g) was washed with water, methanol(2×) and water and then suction dried. The foam residue from thehydrolysis reaction was dissolved in water (100 mL) and treated with thewashed Dowex resin (75 g), and stirred for 60 min, until a sample of theaqueous solution no longer tested positive with ninhydrin stain. Themixture was filtered and the resin washed successively with water,methanol, methylene chloride, methanol, methylene chloride, methanol,and finally water and suction dried briefly.

The resin was then stirred with 2N hydrochloric acid (50 mL) for 15 minand the aqueous decanted into a fritted funnel/filter flask and saved.This was repeated three times with 2N hydrochloric acid (3×50 mL), andthe last resin stir was filtered and rinsed with water (20 mL). Thecombined aqueous filtrate was concentrated in vacuo and the residualfoam dissolved three times in water (20 mL) and concentrated in order toremove residual HCl.

The off-white foamy solid was then dissolved in 30 mL water, frozen at−78° C. and lyophilized to dryness (36 hrs) to afford the product,(3R,4S)-3-amino-1-((S)-2-aminopropanyoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylicacid, dihydrochloride salt (10) as its dihydrochloride salt, as anoff-white powder (4.90 g, 84% over 2 steps). The final compound wasobtained as a 3:2 mixture of rotamers, at room temperature. ¹H-NMR (D₂O,400 MHz): δ_(H): 4.16-4.04 (1.6H, m), 3.95 (0.4H, m), 3.85 (0.6H, m),3.68 (0.4H, m), 3.47-3.35 (1.6H, m), 3.18 (0.4H, m), 2.58 (0.6H, m) and2.47 (0.4H, m), 1.52 (1H, m), 1.38 (1.2H, d, J=7.3 Hz) and 1.34 (1.8H,d, J=7.0 Hz), 1.32-1.09 (3H, m) and 0.64 (2H, m). LC-MS: ESI+ (0.1%HCOOH in IPA/water): m/z for C₁₁H₂₂BN₃O₅: expected 287.2, observed 288.2(M+H)⁺, 270.2 (M+H−H₂O)⁺, 252.2 (M+H−2H₂O)⁺, ESI−: 268.2 (M−H—H₂O)⁻.

Synthesis of(3R,4S)-3-amino-1-((S)-2-aminopropanoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylicacid (13)

Step 1: Synthesis of (3R,4S)-benzyl-1-((S)-2-aminopropanoyl)-3-azido-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-3-carboxylate,TFA salt (11)

A solution of (3R,4S)-benzyl-3-azido-1-((S)-2-((tert-butoxycarbonyl)amino)propanoyl)-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-3-carboxylate(30.04 g, 51.31 mmol) in anhydrous dichloromethane (250 mL) was cooledto 0° C. and then a solution of TFA (50 mL) in dichloromethane (50 mL)was added drop wise over 10 minutes. The solution was allowed to warm toroom temperature and then stirred at this temperature for 3 hours, untilTLC showed complete consumption of the starting material. The reactionmixture was concentrated in vacuo to give a pale yellow oil. This oilwas dissolved in toluene (100 mL) and concentrated. The azeotropicprocedure was repeated three times, to give the product (11), as the TFAsalt, (30.85 g) as a pale yellow oil. ¹H-NMR (400 MHz, D4-MeOH) δ: 7.39(4H, m), 7.15 (1H, m), 5.29 (2H, dd, J=14, 12 Hz), 4.25-3.20 (5H, m),2.51 (1H, m), 1.50-1.25 (6H, including 1.47 (1.5H, d, J=7.0 Hz) and 1.31(1.5H, d, J=6.9 Hz (alanine rotamers))), 1.20 (12H, s)), 1.07 (1H, m)and 0.65 (2H, m). LCMS (ESI +ve): C₂₄H₃₆BN₅O₅ m/z calculated 485.3,found 486.2 (MH⁺).

Step 2: synthesis of (3-((3S,4R)-1-((S)-2-aminopropanoyl)-4-azido-4-((benxyloxy)carbonyl)pyrrolodin-3-yl)propyl)boronicacid, hydrochloride salt

The TFA salt of (3R,4S)-benzyl-1-((S)-2-aminopropanoyl)-3-azido-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-3-carboxylate(30.76 g, 51.31 mmol), was dissolved in a biphasic mixture of methanol(200 mL) and hexane (400 mL). Isobutylboronic acid (18.31 g, 179.6 mmol)and then 2N Hydrochloric acid (50.85 mL, 101.7 mmol) was added. Thereaction mixture was stirred vigorously at room temperature for 16hours. The methanol phase was separated and washed with hexane (5×100mL) and then concentrated in vacuo to give the boronic acid, as thehydrochloride salt, as an off-white foam. ¹H-NMR (400 MHz, D₂O) δ:7.48-7.42 (5H, m), 5.31 (2H, m), 4.22 (1H, dd, J=13, 6.5 Hz), 3.95-3.10(4H, m), 2.71-2.51 (1H, m), 1.40-1.25 (3H, m), 1.25-0.98 (4H, mincluding 1.20 (1.5H, d, J=6.9 Hz) and 1.07 (1.5H, d, J=6.9 Hz (alaninerotamers))) and 0.69 (2H, m). LCMS (ESI +ve): C₁₈H₂₆BN₅O₅ m/z calculated403.2, found 404.2 (MH⁺).

Step 3: synthesis of (3-((3S,4R)-1-((S)-2-aminopropanoyl)-4-azido-4-((benxyloxy)carbonyl)pyrrolodin-3-yl)propyl)boronicacid (12)

The hydrochloride salt of (3-((3S,4R)-1-((S)-2-aminopropanoyl)-4-azido-4-((benxyloxy)carbonyl)pyrrolodin-3-yl)propyl)boronicacid, from the previous step, was dissolved in 30 mL water and then thepH of the solution was adjusted to pH 9 by the careful addition of solidpotassium carbonate. The resultant solution was saturated with theaddition of solid sodium chloride and then was extracted withdichloromethane (5×100 mL). The combined dichloromethane phase was driedover magnesium sulfate, filtered and concentrated in vacuo to give theproduct (12), as its free base, as a white foamy solid (19.4 g, 48.11mmol, 94%). ¹H-NMR (400 MHz, D4-MeOH) δ: 7.44-7.36 (5H, m), 5.31 (1H, d,J=1.8 Hz), 5.27 (1H, d, J=1.8 Hz) 4.05 (1H, dd, J=12, 5 Hz), 3.80 (1H,m), 3.69-3.55 (2H, m), 3.45-3.30 (1H, m), 2.51 (1H, m), 1.40-1.05 (7H,m, including 1.22 (1.5H, d, J=6.8 Hz) and 1.07 (1.5H, d, J=6.8 Hz(alanine rotamers))) and 0.63 (2H, m). LCMS (ESI +ve): C₁₈H₂₆BN₅O₅ m/zcalculated 403.2, found 404.7 (MH⁺).

Step 4: synthesis of (3R,4S)-3-amino-1-((S)-2-aminopropanoyl)-4-(3-boronopropyl)-yl)pyrrolidine-3-carboxylate(13)

The azido benzyl ester, (3-((3S,4R)-1-((S)-2-aminopropanoyl)-4-azido-4-((benxyloxy)carbonyl)pyrrolodin-3-yl)propyl)boronicacid (9.70 g, 24.06 mmol) was suspended in a mixture of water (300 mL)and ethyl acetate (30 mL) and stirred vigorously. 10% Palladium oncharcoal (2.6 g, 0.1 eq) was added and then the stirred mixture wasevacuated under mild vacuum, and flushed with hydrogen. Theevacuation/flushing procedure was repeated 3× to remove air and exchangeit with hydrogen and then the reaction was stirred vigorously overnightat room temperature under a hydrogen balloon, at which time, LCMSanalysis of a filtered aliquot showed the complete reduction of theazide and benzyl ester groups. The reaction mixture was put under vacuumto remove hydrogen and then flushed with nitrogen, filtered through apad of celite (with 3 water washes) and then the solution wasconcentrated to approx 50 mL in vacuo. The resultant aqueous solutionwas filtered through a 4 micron filter (to remove trace Pd) and thenconcentrated in vacuo to give the title compound (13) as a white powder(6.45 g, 93%). ¹H-NMR (400 MHz, D₂O) δ: 4.12 (1H, m), 4.05 (1H, m), 3.92(1H, m), 3.60-3.22 (2H, m), 2.47-2.18 (1H, m), 1.58-1.31 (6H, mincluding 1.46 (3H, d, J=6.9 Hz)), 1.24-1.19 (1H, m) and 0.79 (2H, m).LCMS (ESI +ve): C₁₁H₂₀BN₃O₅ m/z calculated 287.2, found 269.9 (MH⁺−H₂O),251.9 (MH⁺−2H₂O) and (ESI −ve): C₁₁H₂₀BN₃O₅ m/z calculated 287.2, found267.7 (M−H—H₂O).

(3R,4S)-3-Amino-1-((S)-2-amino-3-methylbutanoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylicacid, dihydrochloride (14)

(3R,4S)-3-amino-1-((S)-2-amino-3-methylbutanoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylicacid, dihydrochloride was prepared in a manner analogous to that setforth in the procedure for compound 10, except(tert-butoxycarbonyl)-L-valine was used as the carboxylic acid in thereaction with 7. The final compound was obtained as a mixture ofrotamers, at room temperature. ¹H-NMR (D₂O, 400 MHz): δ_(H): 4.10 (1H,m), 3.96-3.87 (2H, m), 3.42-3.36 (1H, m), 3.07-2.91 (1H, m), 2.55 (0.7H,m) and 2.40 (0.3H, m), 2.11 (1H, m), 1.51 (1H, m), 1.34-1.10 (3H, m),0.92 (3H, d, J=6.9 Hz), 0.87 (3H, d, J=6.9 Hz), 0.65 (2H, m). LC-MS:ESI+ (0.1% HCOOH in IPA/water): m/z for C₁₁H₂₆BN₃O₅: expected 315.2,observed 326.3 (M+H+HCOOH-H₂O)⁺, 298.3 (M+H−H₂O)⁺, 280.3 (M+H−2H₂O)⁺,ESI−: 296.2 (M−H—H₂O)⁻.

(3R,4S)-3-Amino-1-((S)-2-amino-3-hydroxypropanoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylicacid, dihydrochloride (15)

(3R,4S)-3-amino-1-((S)-2-amino-3-hydroxypropanoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylicacid, dihydrochloride was prepared in a manner analogous to that setforth in the procedure for compound 10, except(S)-3-(tert-butoxycarbonyl)-2,2-dimethyloxazolidine-4-carboxylic acidwas used as the carboxylic acid in the reaction with 7. The finalcompound was isolated as a 2:1 mixture of rotamers at room temperature.¹H-NMR (D₂O, 400 MHz): δ_(H): 4.21 (1H, m), 4.11 (1H, d, J=13.0 Hz),3.93 (1H, dd, J=11.5, 8.6 Hz), 3.86-3.74 (2H, m), 3.47 (1H, m),3.04-2.96 (1H, m), 2.56 (0.7H, m) and 2.44 (0.3H, m), 1.51 (1H, m),1.29-1.12 (3H, m), 0.64 (2H, m). LC-MS: ESI+ (0.1% HCOOH in IPA/water):m/z for C₁₁H₂₂BN₃O₆: expected 303.16, observed 314.2 (M+H+HCOOH-H₂O)⁺,286.2 (M+H−H₂O)⁺, 268.2 (M+H−2H₂O)⁺, ESI−: 284.1 (M−H—H₂O)⁻.

trans-3-amino-1-((S)-2-amino-3-(1H-imidazol-4-yl)propanoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylicacid, trihydrochloride (16)

trans-3-amino-1-((S)-2-amino-3-(1H-imidazol-4-yl)propanoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylicacid, trihydrochloride was prepared in a manner analogous to that setforth in the procedure for compound 10, except(tert-butoxycarbonyl)-L-histidine was used as the carboxylic acid, andracemic 5 was used instead of 5a. ¹H-NMR (D₂O, 400 MHz): δ_(H): 8.57(1H, d, J=9.0 Hz), 7.33 (1H, d, J=16.9 Hz), 4.20-3.70 (3H, m), 3.51 (1H,m), 3.37-3.24 (3H, m), 2.58 (1H, m), 1.50 (1H, m), 1.39-1.11 (3H, m) and0.68 (2H, m). LC-MS: ESI+ (0.1% HCOOH in IPA/water): m/z forC₁₄H₂₄BN₅O₅: expected 353.18, observed 354.41 (M+H)⁺, 336.44 (M+H−H₂O)⁺,318.49 (M+H−2H₂O)⁺.

(3R,4S)-3-amino-4-(3-boronopropyl)-1-glycylpyrrolidine-3-carboxylic acid(17)

(3R,4S)-3-amino-4-(3-boronopropyl)-1-glycylpyrrolidine-3-carboxylic acidwas prepared in a manner analogous to that set forth in the procedurefor compound 13, except (tert-butoxycarbonyl)glycine was used as thecarboxylic acid in the reaction with 7. The final compound was isolatedas a 3:2 mixture of rotamers at room temperature. ¹H-NMR (D₂O, 400 MHz):δ_(H): 4.08-3.83 (2H, m), 3.91 (2H, d, J=4.6 Hz), 3.63-3.53 (1H, m),3.40-3.22 (1H, m), 2.57-2.37 (1H, m), 1.61 (1H, m), 1.50-1.35 (2H, m),1.25 (1H, m) and 0.78 (2H, m). LC-MS: ESI+ (0.1% HCOOH in IPA/water):m/z for C₁₀H₂₀BN₃O₅: expected 273.15, observed 256.2 (M+H—H₂O)⁺, 238.2(M+H−2H₂O)⁺; ESI−: 254.2 (M−H—H₂O)⁻.

(3R,4S)-3-amino-1-(2-amino-2-methylpropanoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylicacid (18)

(3R,4S)-3-amino-1-(2-amino-2-methylpropanoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylicacid was prepared in a manner analogous to that set forth in theprocedure for compound 13, except2-((tert-butoxycarbonyl)amino)-2-methylpropanoic acid was used as thecarboxylic acid in the reaction with 7. The final compound was isolatedas a 2.1 mixture of rotamers at room temperature. ¹H-NMR (D₂O, 400 MHz):δ_(H): 4.38-3.88 (2H, m), 3.72-3.63 (1H, m), 3.40-3.08 (1H, m),2.75-2.52 (1H, m), 1.71 and 1.69 (4H, s and 2H, s, CMe₂ 2:1 rotamers),1.64 (1H, m), 1.55-1.41 (2H, m), 1.31 (1H, m) and 0.81 (2H, m). LC-MS:ESI+ (0.1% HCOOH in IPA/water): m/z for C₁₂H₂₄BN₃O₅: expected 301.18,observed 284.0 (M+H−H₂O)⁺, 266.0 (M+H−2H₂O)⁺, ESI−: 281.8 (M−H—H₂O)⁻.

(3R,4S)-3-amino-1-((S)-2-aminobutanoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylicacid (19)

(3R,4S)-3-amino-1-((S)-2-aminobutanoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylicacid was prepared in a manner analogous to that set forth in theprocedure for compound 13, except(S)-2-((tert-butoxycarbonyl)amino)butanoic acid was used as thecarboxylic acid in the reaction with 7. The final compound was isolatedas a 2:1 mixture of rotamers at room temperature. ¹H-NMR (D₂O, 400 MHz):δ_(H): 4.07-3.87 (3H, m), 3.62-3.27 (2H, m), 2.45-2.17 (1H, m), 1.80(2H, m), 1.58 (1H, m), 1.50-1.33 (2H, m), 1.21 (1H, m), 0.99 (3H, m) and0.79 (2H, m). LC-MS: ESI+ (0.1% HCOOH in IPA/water): m/z forC₁₂H₂₄BN₃O₅: expected 301.18, observed 284.2 (M+H−H₂O)⁺; ESI−: 282.4(M−H—H₂O)⁻.

(3R,4S)-3-amino-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylicacid (20)

(3R,4S)-3-amino-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylicacid was prepared in a manner analogous to that set forth in theprocedure for compound 13, except(S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoic acid was used asthe carboxylic acid in the reaction with 7. The final compound wasisolated as a 2:1 mixture of rotamers at room temperature. ¹H-NMR (D₂O,400 MHz): δ_(H) 4.21-3.92 (2H, m), 3.81 [(0.67H, s) and 3.71 (0.33H, s)2:1 rotamers CHN], 3.66-3.33 (2H, m), 2.47-2.17 (1H, m), 1.59 (1H, m),1.51-1.35 (2H, m), 1.23 (1H, m), 1.06 and 1.04 [(6H, s) and (3H, s) tBu2:1 rotamers) and 0.81 (2H, m). LC-MS: ESI+(0.1% HCOOH in IPA/water):m/z for C₁₄H₂₈BN₃O₅: expected 329.21, observed 312.4 (M+H−H₂O)⁺, 294.4(M+H−2H₂O)⁺, ESI−: 310.4 (M−H—H₂O)⁻.

(3R,4S)-3-amino-1-(1-aminocyclopropane-1-carbonyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylicacid (21)

(3R,4S)-3-amino-1-(1-aminocyclopropane-1-carbonyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylicacid was prepared in a manner analogous to that set forth in theprocedure for compound 13, except(S)-2-((tert-butoxycarbonyl)amino)-2-cyclopropylacetic acid was used asthe carboxylic acid in the reaction with 7. The final compound wasisolated as a 3:2 mixture of rotamers at room temperature. ¹H-NMR (D₂O,400 MHz): δ_(H): 4.37-3.99 (2H, m), 3.85-3.30 (2H, m), 2.54-2.38 (1H,m), 1.61 (1H, m), 1.47-1.33 (2H, m), 1.24 (1H, m), 1.09 (1H, m) 0.97(1H, m), 0.89 (2H, m) and 0.81 (2H, m). LC-MS: ESI+ (0.1% HCOOH inIPA/water): m/z for C₁₂H₂₂BN₃O₅: expected 299.17, observed 282.1(M+H−H₂O)⁺, 264.1 (M+H−2H₂O)⁺, ESI−: 280.2 (M−H—H₂O)⁻.

Example 2: Oral Bioavailability Studies

Compound dosing solutions were prepared at 2.5 and 5 mg/mL in water.Female C57BL/6 mice (16-20 g) from Charles River Laboratories(Hollister, Calif.) were housed in cages for at least 3 days prior todosing. PicoLab 5053 irradiated rodent diet was provided ad libitumthroughout the study. Compounds were administered once to theappropriate animals by oral gavage at either 25 or 50 mg/kg (10 mL/kg).Blood samples were collected (3 animals per time point) at 30 min and 1,2, 4, 8 hr post-dose for the 25 mg/kg studies, and at 1 hour for the 50mg/kg studies. The blood samples were maintained on wet ice and thencentrifuged for 10 min in a refrigerated centrifuge. The resultantplasma was separated, transferred to labeled polypropylene tubes andstored frozen in a freezer set to maintain under −70° C. until analysis.

The plasma samples were analyzed by an LC-MS system. 50 μL of a plasmasample was mixed with 100 μL of acetonitrile/water (80:20) with 0.1% TFAcontaining 100 ng/mL of an internal standard. The mixture was vortexedand centrifuged. 30 μL of the supernatant was transferred to a 96-wellplate containing 90 μL of water with 0.1% formic acid. 20 μL of theresulting solution was injected into a SCIEX QTRAP4000 LC/MS/MS equippedwith an electrospray ionization source for quantification.

Oral PK parameters were calculated by noncompartmental analysis of theconcentration-time data using Phoenix WinNonLin 6.3 software (Pharsight,Mountain View, Calif.). Area under the concentration-time curve (AUC)was estimated using a linear-up and log-down trapezoidal method,calculated from the dosing time to the last measurable concentration.

AUC for exemplary compounds is shown below:

As compared to the proline, trifluoromethyl phenylalanine, andN-methylphenylalanine-derived compounds, the oral exposure for thealanine, valine, and serine derivatives are more favorable.

Example 3: Pharmacokinetic Studies

The pharmacokinetics of the compounds of the invention were studiedafter administration of a single dose (50 mg/kg) at a single time point(1 hour) in mice. Plasma concentrations were determined as described inExample 2. Results for exemplary compounds are shown below:

Example 4: Single-Agent Anti-Tumor Activity of Compound 10 Lewis LungCarcinoma Efficacy Study

Female C57.Bl/6 mice (n=40) were implanted subcutaneously with 1×10⁶Lewis Lung Carcinoma cells suspended in PBS. The day followingimplantation, mice were randomized into 4 groups of n=10 mice to receivethe following treatments dosed orally twice daily until study end: 1)Vehicle (water); 2) Compound 10 at 50 mg/kg formulated in water; 3)Compound 10 at 100 mg/kg formulated in water; or 4) Compound 10 at 200mg/kg formulated in water. Tumors were measured three times per weekwith digital calipers and tumor volumes calculated with the followingformula: tumor volume (mm³)=(a×b²/2) where ‘b’ is the smallest diameterand ‘a’ is the largest perpendicular diameter. ***P-value <0.001,****P-value <0.0001 (Two-sided T-test). Results are shown in FIG. 1.

Madison109 Efficacy Study

Female balb/c mice (n=20) were implanted subcutaneously with 5×10⁴Madison109 murine lung carcinoma cells suspended in PBS. The dayfollowing implantation, mice were randomized into 2 groups of n=10 miceto receive the following treatments dosed orally twice daily until studyend: 1) Vehicle (water); or 2) Compound 10 at 100 mg/kg formulated inwater. Tumors were measured three times per week with digital calipersand tumor volumes calculated with the following formula: tumor volume(mm³)=(a×b²/2) where ‘b’ is the smallest diameter and ‘a’ is the largestperpendicular diameter. *P-value <0.05 (Two-sided T-test). Results areshown in FIG. 2.

B16 Efficacy Study

Female C57.Bl/6 mice (n=20) were implanted subcutaneously with 2×10⁶B16F10 murine melanoma cells suspended in PBS. The day followingimplantation, mice were randomized into 2 groups of n=10 mice to receivethe following treatments dosed orally twice daily until study end: 1)Vehicle (water); or 2) Compound 10 at 100 mg/kg formulated in water.Tumors were measured three times per week with digital calipers andtumor volumes calculated with the following formula: tumor volume(mm³)=(a×b²/2) where ‘b’ is the smallest diameter and ‘a’ is the largestperpendicular diameter. ***P-value <0.001 (Two-sided T-test). Resultsare shown in FIG. 3.

Example 5: 4T1 Combination Therapy Studies

Female balb/c mice (n=40) were implanted in the mammary fat pad with1×10⁵ 4T1 murine mammary carcinoma cells suspended in PBS. The dayfollowing implantation, mice were randomized into 4 groups of n=10 miceeach to receive the following treatments: 1) Vehicle (water) dosedorally twice daily until study end; 2) Compound 10 at 100 mg/kgformulated in water dosed orally twice daily until study end; 3) Thecombination of anti-PD-1 (clone RMPI-14) dosed IP at 5 mg/kg on days 3,6, and 9 post-implant plus anti-CTLA-4 (clone 9H10) dosed IP at 5 mg/kgon days 2, 5, and 8 post-dose; or 4) the triple combination of compound10 plus anti-PD-1 plus anti-CTLA-4 at their respective regimens. Tumorswere measured three times per week with digital calipers and tumorvolumes calculated with the following formula: tumor volume(mm³)=(a×b²/2) where ‘b’ is the smallest diameter and ‘a’ is the largestperpendicular diameter. ***P-value <0.001 (Two-sided T-test). On day 25,mice were sacrificed and lungs perfused with India Ink (25% in PBS) thenharvested and fixed in 100% ethanol: 10% neutral buffered formalin:acetic acid mixture at 10:1:0.5 ratio. The number of lung metastases wascounted manually in a blinded manner. Results are shown in FIG. 4.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

We claim:
 1. A compound having a structure of formula (I):

or a pharmaceutically acceptable salt or prodrug thereof; wherein: R^(a)is H or is selected from optionally substituted alkyl, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, (cycloalkyl)alkyl,(heterocycloalkyl)alkyl, aryl, heteroaryl, aralkyl, and heteroaralkyl;R^(b) is H or is selected from optionally substituted alkyl, alkenyl,alkynyl, acyl, —C(O)O(alkyl), and —C(O)O(aryl); each R^(c) isindependently selected from H or alkyl, or two occurrences of R^(c) aretaken together with the intervening —O—B—O— atoms to form an optionallysubstituted boron-containing ring; X is O or S; R¹ and R² are eachindependently selected from H and optionally substituted alkyl, alkenyl,alkynyl, cycloalkyl, (cycloalkyl)alkyl, heterocycloalkyl,(heterocycloalkyl)alkyl, aryl, heteroaryl, aralkyl, and heteroaralkyl;R¹ and R² are taken together with the intervening atoms to form anoptionally substituted 5- to 7-membered ring; and R³ is H or optionallysubstituted alkyl; or R¹ and R³ are taken together with the interveningatoms to form an optionally substituted 5- to 7-membered ring; whereinthe compound is not:


2. The compound of claim 1, having a structure of formula (Ia):


3. The compound of claim 1, having a structure of formula (Ib):


4. The compound of claim 1, having a structure of formula (Ic):


5. The compound of claim 1, having a structure of formula (Id):


6. The compound of claim 1, having a structure of formula (Ie):


7. The compound of claim 1, having a structure of formula (If):


8. The compound of claim 1, having a structure of formula (Ig):


9. The compound of claim 1, having a structure of formula (Ih):


10. The compound of any one of claims 1-3, wherein R² is H.
 11. Thecompound of any one of claims 1-10, wherein R^(a) is H or optionallysubstituted alkyl.
 12. The compound of claim 11, wherein R^(a) is H. 13.The compound of any one of claims 1-12, wherein R^(b) is H or optionallysubstituted alkyl or acyl.
 14. The compound of claim 13, wherein R^(b)is H.
 15. The compound of any one of claims 1-14, wherein, for eachoccurrence, R^(c) is H.
 16. The compound of any one of claims 1-14,wherein two occurrences of R^(c) are taken together to form anoptionally substituted dioxaborolane, dioxaborolanone,dioxaborolandione, dioxaborinane, dioxaborinanone, or dioxaborinandione.17. The compound of any one of claims 1-15, wherein X is O.
 18. Thecompound of any one of claims 1-17, wherein if R¹ is H, then R³ is notbenzyl.
 19. The compound of any one of claims 1-18, wherein R¹ is H. 20.The compound of any one of claims 1-18, wherein if R¹ is benzyl, then R³is not methyl.
 21. The compound of any one of claims 1-17 and 20,wherein R¹ is optionally substituted aralkyl, heteroaralkyl,(cycloalkyl)alkyl, or (heterocycloalkyl)alkyl.
 22. The compound of anyone of claims 1-17 and 20, wherein R¹ is optionally substituted aralkylor heteroaralkyl.
 23. The compound of claim 22, wherein R¹ is benzyl.24. The compound of claim 22, wherein R¹ is not benzyl substituted by—CF₃.
 25. The compound of claim 22, wherein R¹ is heteroaralkyl, such as—CH₂-(1H-imidazol-4-yl).
 26. The compound of any one of claims 1-17,wherein R¹ is optionally substituted alkyl, alkenyl, or alkynyl.
 27. Thecompound of claim 26, wherein R¹ is alkyl, optionally substituted by oneor more substituents independently selected from hydroxy, halo,haloalkyl, alkoxy, —SH, —S-(alkyl), —SeH, —Se-(alkyl), aryl, heteroaryl,cycloalkyl, heterocycloalkyl, amino, carboxylic acid, ester, guanidino,and amido.
 28. The compound of claim 27, wherein R¹ is alkyl, optionallysubstituted by one or more substituents independently selected fromhydroxy, halo, haloalkyl, alkoxy, —SH, —S-(alkyl), —SeH, —Se-(alkyl),heteroaryl, cycloalkyl, heterocycloalkyl, amino, carboxylic acid, ester,guanidino, and amido.
 29. The compound of claim 28, wherein R¹ is alkyl,optionally substituted by one or more substituents independentlyselected from hydroxy, alkoxy, haloalkyl, and —S-(alkyl).
 30. Thecompound of any one of claims 1-17, wherein R¹ is selected fromoptionally substituted cycloalkyl, heterocycloalkyl, aryl, andheteroaryl.
 31. The compound of any one of claims 1-17, wherein R¹ is anamino acid side chain of Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln,Cys, Sec, Gly, Ala, Val, Ile, Leu, Met, Phe, Tyr, or Trp.
 32. Thecompound of any one of claims 1-31, wherein R³ is H.
 33. The compound ofany one of claims 1-17, wherein R¹ and R³ are taken together with theintervening atoms to form a substituted 5-membered ring.
 34. Thecompound of any one of claims 1-17, wherein R¹ and R³ are taken togetherwith the intervening atoms to form an optionally substituted 6- or7-membered ring.
 35. The compound of claim 34, wherein R¹ and R³, takentogether with the intervening atoms, do not form atetrahydroisoquinolinyl ring.
 36. The compound of claim 1, having astructure selected from:

or a pharmaceutically acceptable salt or prodrug thereof.
 37. Apharmaceutical composition comprising a compound of any one of claims1-36 and a pharmaceutically acceptable carrier.
 38. A method of treatingor preventing cancer, comprising administering to a subject in needthereof a therapeutically effective amount of a compound of any one ofclaims 1-36 or a pharmaceutical composition of claim
 37. 39. The methodof claim 38, wherein the cancer is Acute Lymphoblastic Leukemia (ALL),Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, Anal Cancer,Appendix Cancer, Atypical Teratoid/Rhabdoid Tumor, Basal Cell Carcinoma,Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Tumor, Astrocytoma,Brain and Spinal Cord Tumor, Brain Stem Glioma, Central Nervous SystemAtypical Teratoid/Rhabdoid Tumor, Central Nervous System EmbryonalTumors, Breast Cancer, Bronchial Tumors, Burkitt Lymphoma, CarcinoidTumor, Carcinoma of Unknown Primary, Central Nervous System Cancer,Cervical Cancer, Childhood Cancers, Chordoma, Chronic LymphocyticLeukemia (CLL), Chronic Myelogenous Leukemia (CML), ChronicMyeloproliferative Disorders, Colon Cancer, Colorectal Cancer,Craniopharyngioma, Cutaneous T-Cell Lymphoma, Ductal Carcinoma In Situ(DCIS), Embryonal Tumors, Endometrial Cancer, Ependymoblastoma,Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, Ewing Sarcoma,Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, ExtrahepaticBile Duct Cancer, Eye Cancer, Fibrous Histiocytoma of Bone, GallbladderCancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor,Gastrointestinal Stromal Tumors (GIST), Germ Cell Tumor, ExtracranialGerm Cell Tumor, Extragonadal Germ Cell Tumor, Ovarian Germ Cell Tumor,Gestational Trophoblastic Tumor, Glioma, Hairy Cell Leukemia, Head andNeck Cancer, Heart Cancer, Hepatocellular Cancer, Histiocytosis,Langerhans Cell Cancer, Hodgkin Lymphoma, Hypopharyngeal Cancer,Intraocular Melanoma, Islet Cell Tumors, Kaposi Sarcoma, Kidney Cancer,Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia, Lip and OralCavity Cancer, Liver Cancer, Lobular Carcinoma In Situ (LCIS), LungCancer, Lymphoma, AIDS-Related Lymphoma, Macroglobulinemia, Male BreastCancer, Medulloblastoma, Medulloepithelioma, Melanoma, Merkel CellCarcinoma, Malignant Mesothelioma, Metastatic Squamous Neck Cancer withOccult Primary, Midline Tract Carcinoma Involving NUT Gene, MouthCancer, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma/PlasmaCell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndrome,Myelodysplastic/Myeloproliferative Neoplasm, Chronic MyelogenousLeukemia (CML), Acute Myeloid Leukemia (AML), Myeloma, Multiple Myeloma,Chronic Myeloproliferative Disorder, Nasal Cavity Cancer, ParanasalSinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-HodgkinLymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Oral Cavity Cancer,Lip Cancer, Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer,Pancreatic Cancer, Papillomatosis, Paraganglioma, Paranasal SinusCancer, Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer,Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumors ofIntermediate Differentiation, Pineoblastoma, Pituitary Tumor, PlasmaCell Neoplasm, Pleuropulmonary Blastoma, Breast Cancer, Primary CentralNervous System (CNS) Lymphoma, Prostate Cancer, Rectal Cancer, RenalCell Cancer, Renal Pelvis Cancer, Ureter Cancer, Transitional CellCancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,Sarcoma, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, SmallIntestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma, SquamousNeck Cancer with Occult Primary, Stomach Cancer, SupratentorialPrimitive Neuroectodermal Tumors, T-Cell Lymphoma, Testicular Cancer,Throat Cancer, Thymoma, Thymic Carcinoma, Thyroid Cancer, TransitionalCell Cancer of the Renal Pelvis and Ureter, Gestational TrophoblasticTumor, Unknown Primary, Unusual Cancer of Childhood, Urethral Cancer,Uterine Cancer, Uterine Sarcoma, Waldenström Macroglobulinemia, or WilmsTumor.
 40. The method of claim 39, wherein the cancer is selected fromacute myeloid leukemia (AML), bladder cancer, breast cancer, colorectalcancer, chronic myelogenous leukemia (CML), esophageal cancer, gastriccancer, lung cancer, melanoma, mesothelioma, non-small cell lungcarcinoma (NSCLC), ovarian cancer, pancreatic cancer, prostate cancer,renal cancer, and skin cancer.
 41. The method of any one of claims38-40, further comprising conjointly administering one or moreadditional chemotherapeutic agents.
 42. The method of claim 41, whereinthe one or more additional chemotherapeutic agents includesaminoglutethimide, amsacrine, anastrozole, asparaginase, AZD5363,Bacillus Calmette-Guérin vaccine (bcg), bicalutamide, bleomycin,bortezomib, buserelin, busulfan, campothecin, capecitabine, carboplatin,carfilzomib, carmustine, chlorambucil, chloroquine, cisplatin,cladribine, clodronate, cobimetinib, colchicine, cyclophosphamide,cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin,demethoxyviridin, dexamethasone, dichloroacetate, dienestrol,diethylstilbestrol, docetaxel, doxorubicin, epirubicin, erlotinib,estradiol, estramustine, etoposide, everolimus, exemestane, filgrastim,fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide,gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide,imatinib, interferon, irinotecan, lenalidomide, letrozole, leucovorin,leuprolide, levamisole, lomustine, lonidamine, mechlorethamine,medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna,metformin, methotrexate, miltefosine, mitomycin, mitotane, mitoxantrone,MK-2206, nilutamide, nocodazole, octreotide, olaparib, oxaliplatin,paclitaxel, pamidronate, pazopanib, pentostatin, perifosine, plicamycin,pomalidomide, porfimer, procarbazine, raltitrexed, rituximab, rucaparib,selumetinib, sorafenib, streptozocin, sunitinib, suramin, talazoparib,tamoxifen, temozolomide, temsirolimus, teniposide, testosterone,thalidomide, thioguanine, thiotepa, titanocene dichloride, topotecan,trametinib, trastuzumab, tretinoin, veliparib, vinblastine, vincristine,vindesine, or vinorelbine.
 43. The method of claim 41, wherein the oneor more additional chemotherapeutic agents includes abagovomab,adecatumumab, afutuzumab, alemtuzumab, anatumomab mafenatox, apolizumab,atezolizumab, avelumab, blinatumomab, BMS-936559, catumaxomab,durvalumab, epacadostat, epratuzumab, indoximod, inotuzumab ozogamicin,intelumumab, ipilimumab, isatuximab, lambrolizumab, MED14736, MPDL3280A,nivolumab, ocaratuzumab, ofatumumab, olatatumab, pembrolizumab,pidilizumab, rituximab, ticilimumab, samalizumab, or tremelimumab. 44.The method of claim 41, wherein the one or more additionalchemotherapeutic agents includes abagovomab, adecatumumab, afutuzumab,anatumomab mafenatox, apolizumab, blinatumomab, catumaxomab, durvalumab,epratuzumab, inotuzumab ozogamicin, intelumumab, ipilimumab, isatuximab,lambrolizumab, nivolumab, ocaratuzumab, olatatumab, pembrolizumab,pidilizumab, ticilimumab, samalizumab, or tremelimumab.
 45. The methodof claim 44, wherein the one or more additional chemotherapeutic agentsincludes ipilimumab, nivolumab, pembrolizumab, or pidilizumab.
 46. Themethod of any one of claims 38-45, wherein the method further comprisesadministering one or more non-chemical methods of cancer treatment, suchas radiation therapy, surgery, thermoablation, focused ultrasoundtherapy, cryotherapy, or a combination of the foregoing.